Team:Freiburg Bioware/NoteBook/Labjournal/September

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September

107. labday 01.09.2010

Loop insertion BioBricks: Sequencing results

Investigator: Achim, Anna

Comment: Sequencing results of Loop insertion BioBricks from 31.08.10; the following clones were sent for sequencing:

2: 453 BAP: No Insert

3:453 His: Insert okay

5: 453 RGD: Mutation in 453 upstream region

7: 587 BAP: Mutation in Bap insert

9:587 His: Insert okay

11:587 KO BAP : Insert okay

13:587 KO empty : Insert okay

16: 587 KO his: Mutation in 587 KO

17: 587 KO rgd: No insert

19 :587 RGD: Insert okay

25: 587 Z34C: Mutation in downstream 587 region

Mini-Preps:

New Mini-Preps of the samples with no/mutated insert were prepared. The following clones were used:

Components	453 BAP	587 BAP	453 RGD	587 KO RGD	587 KO HIS	453 Z34C	587 Z34C	587 KO Z34C	587 KO Z34C SPACER
Clone	1.3 + 1.4	2.3 + 2.4	4.3 + 4.4	6.3 + 6.4	9.3 + 9.4	11.3 + 11.4	12.2 + 12.4	13.2 + 13.4	14.3 + 14.4

Comment: Samples 1.3, 2.3, 4.3, 6.3, 9.3, 11.3, 12.2, 13.2, 14.3 were sent for sequencing. For sequencing results go to labday 02.09.10.

.

Design of primers for pAAV_RC

Investigator: Bea

Primers for different cloning strategies of the synthesized cap gene into pAAV_Rc have been designed.

The first primers are mutagenesis primers which delete the KpnI recognition site at position 3968. These primers can (or should be used) be used if cloning with BspMI/BfuI (isochizomer) does not cut the pAAV_RC efficiently. Instead of using this enzyme we can use XcmI to subclone the cap gene with performing a site-directed mutagenesis afterwards with designed primers.
The second idea is to design oligoduplexes (hybridized oligos) which contain the recognition site for BspMI. In Gormley et al (2002) the idea was to add oligodupexes with the recognition site for BspMI and therefore provide the "second recognition site" in trans.

Next step: Check primers and order them today! Designed primers are stored in my Workingfolder under paav_rc and a word document was created whcih can be found under Oligos --> primers for different strategies...

Sequencing results of pSB1C3_Zegfr:1907_Linker

Investigator: Hanna

Comment: Sequencing results looked all well. One bp was missing in the theoretical sequence of SEG suffix - but is correct in the actual sequence.

pSB1Cr_Zegfr:1907_LongLinker:

pSB1Cr_Zegfr:1907_SEG:

pSB1Cr_Zegfr:1907_MiddleLinker:

pSB1Cr_Zegfr:1907_ShortLinker:

These constructs will be cloned into pCerulean for N-terminal fusion to VP2.
go to the top ;)

Trafo evalutaion and Inoculation

Investigator: Kira
The plate with transformed CD-biobrick contains around 40 colonies. 4 of them will be inoculated into LB and incubated @37 C over-night.
go to the top ;)

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

Investigator: Patrick

The two Ligations were performed according to the standard protocol:
1 µl T4 DNA ligase, 1 µl 10x Buffer, 4,6 µl vector (P273), 3,34 µl Insert (P276, upper and lower band).
Incubation time: 50 minutes.

During the transformation i forgot to put the cells into the 37°C shaker so i incubated them afterwards again for 45 minutes at 37 °C on a shaker.

The mutual conles will be picked tomorrow to inoculate 10 ml DYT following a mini-prep on 03.09.2010

Transfection, Seeding AAV293 and HT1080

Investigator: Kerstin

Transfection: 10 plates, same treatment (10µg of RC (P357), pHelper (P356)and mVenus (P263), following the standard protocol)
Seeding HT1080 for transduction with virus from 21.08.2010 (10µg of RC, pHelper, TKGMK; pH=7,10)
Seeding AAV293 for transfection with plasmids without HgH or beta-globin (verifying functionality)

Cloning of Z_EGFR:1907_Middle-Linker,Z_EGFR:1907_Short-Linker, Z_EGFR:1907_SEG-Linker and Z_EGFR:1907_Long-Linker into pCerulean

Investigator: Stefan
Digestion:

components	Vector (P273)	Z_EGFR:1907_Middle-Linker (P290)	Z_EGFR:1907_Short-Linker (P292)	Z_EGFR:1907_SEG-Linker (P296)	Z_EGFR:1907_Long-Linker (P298)
DNA	3,8	8,8	10,2	9,2	8,7
BSA (10x)	2	2	2	2	2
Buffer 4 (10x)	2	2	2	2	2
XbaI	1	1	1	1	1
PstI	1	1	1	1	1
H₂O	10,2	5,2	3,8	4,8	5,3
Total volume	20	20	20	20	20

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 50 minutes

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

P273: c= 3,1 ng/µl
P290: c= 3,2 ng/µl
P292: c= 1,8 ng/µl
P296: c= 3,0 ng/µl
P298: c= 3,2 ng/µl

T4 Ligation:

The Ligation was performed as following:

Comment: No sufficent vector concentration, therefore 5 µl of vector was used for every approach.

Vector Volume: 5 µl
Insert Volume: 3 µl

1µl T4-Ligase buffer (10x)
8µl (Vector + Insert) mix
1µl T4-Ligase

Incubating for 40 minutes.

Transformation:

Trafo was performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol

108. labday 02.09.2010

Sequencing of loop insertions BioBricks: 2nd round

Investigator: Achim, Anna

We analyzed the new sequencing results and found two correct sequences:

587 BAP clone 2.3
453 RGD clone 4.3

The other samples didn't contain any inserts or had wrong insertions. Apparently, the vector tends to religate easily. A possible reason for the religation of the incompatible ends is the overnight ligation at 18°C. We sent preps of the remaining 7 ligations for sequencing and will prep new clones or repeat the ligation tomorrow, depending on the new sequences.

Comment: Clones 1.4, 6.4, 9.4, 11.4, 12.4, 13.4, 14.4 were sent for sequencing. For sequencing results go to labday 03.09.10.

.

Mini-preps of pSB1C3_SV40 and pCerulean_VP1_NLS

Investigator: Bea (Chris L)

Mini-Prep was performed according to the standard protocol

P363 = pSB1C3_SV40 clone 1 = 277,70 ng/µl

P364 = pSB1C3_SV40 clone 2 = 265,01 ng/µl

P365 = pCerulean_VP1up_NLS (1) clone 1 = 473,14 ng/µl

P366 = pCerulean_VP1up_NLS (1) clone 2 = 455,88 ng/µl

P367 = pCerulean_VP1up_NLS (1) clone 3 = 482,86 ng/µl

P368 = pCerulean_VP1up_NLS (2) clone 1 = 438,52 ng/µl

P369 = pCerulean_VP1up_NLS (2) clone 2 = 478,00 ng/µl

P370 = pCerulean_VP1up_NLS (2) clone 3 = 419,00 ng/µl
Construct were digested with XbaI and PstI for 45 minutes at 37°C. The loading plan on the 1% agarose gel looks like this:

_M_ ___ _363_ ___ _364_ _365_ _366_ _367_ _368_ _369_ _370_ ___

Results: P365 was sent for sequencing because test digestion looks goos. In contrast to the SV40 appraoch. This needs to be repeated tomorrow.

Plasmid: pCerulean_VP1up_NLS
Plasmid number: P365
Tube name: SB1
Primer used: CMV-F

Mini-Prep and test digestion of pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker

Investigator: Hanna

Comment: Zegfr:1907 (Affibody) and the His-Tag which was coupled to the middle linker were cloned into pCerulean. 3 clones of each construct were picked yesterday.

Plasmid Mini-Prep

new vector name: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker

Glycerol Stocks
1. pCerulean_Zegfr:1907

	Clone 1	Clone 2	Clone 3
Bacteria strain	XL1b	XL1b	XL1b
Plasmidname	pCerulean_Zegfr:1907	pCerulean_Zegfr:1907	pCerulean_Zegfr:1907
Date	2.9.10	2.9.10	2.9.10
given glycerol-stock no.	B275	B276	B277
given plasmid no.	P371	P372	P373

2. pCerulean_6xHis_MiddleLinker

	Clone 1	Clone 2	Clone 3
Bacteria strain	XL1b	XL1b	XL1b
Plasmidname	pCerulean_6xHis_MiddleLinker	pCerulean_6xHis_MiddleLinker	pCerulean_6xHis_MiddleLinker
Date	2.9.10	2.9.10	2.9.10
given glycerol-stock no.	B278	B279	B280
given plasmid no.	P374	P375	P376

Test digestion

buffer used: 4; Restriction-enzymes used: Enzyme 1 PstI-HF ; Enzyme 2 EcoRI-HF
Plasmid
- Given Plasmid-Number: 371; DNA concentration: 381.3 ng/µL;
- Given Plasmid-Number: 372; DNA concentration: 377.5 ng/µL;
- Given Plasmid-Number: 373; DNA concentration: 409.7 ng/µL;
- Given Plasmid-Number: 374; DNA concentration: 404.2 ng/µL;
- Given Plasmid-Number: 375; DNA concentration: 366.1 ng/µL;
- Given Plasmid-Number: 376; DNA concentration: 375.0 ng/µL;

Comments::)

Pipetting scheme for each test digestion:

Components	Volume/µL
DNA	2.7
BSA (10x)	1
Buffer no. 4 (10x)	1
EcoRI-HF	0.5
PstI-HF	0.5
H₂O	4.3
Total volume	10

Incubation: 55 minutes

Agarose-Gel:

0.875 g Agarose, 50 mL TAE (1.75 %), 3 µL EthBr, at 115 Volt, running time: 35 minutes

Sample	Sample/µl]	Loading dye (6x)/µl	Expected size
pCerulean_Zegfr:1907 clone 1	10 µl	2 µl	231 bp
pCerulean_Zegfr:1907 clone 2	10 µl	2 µl	231 bp
pCerulean_Zegfr:1907 clone 3	10 µl	2 µl	231 bp
pCerulean_6xHis_MiddleLinker clone 1	10 µl	2 µl	105 bp
pCerulean_6xHis_MiddleLinker clone 2	10 µl	2 µl	105 bp
pCerulean_6xHis_MiddleLinker clone 3	10 µl	2 µl	105 bp

Marker: GeneRuler ladder mix

	Marker /µL	Sample P371 /µl	Sample P372 /µl	Sample P373 /µl	Sample P374 /µl	Sample P375 /µl	Sample P376 /µl
Lane	5	12	12	12	12	12	12

Comments: Test digestion looked well:

Clone 1 of each construct were sent for sequencing (P371 and P374 = HW1 and HW2). Used primer: GATC_std_CMV-F.

Midi-Prep of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1 & pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1

Investigators: Chris W.

Midi-Preps of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1=P377 and pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1=P378

The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no.	P377	P378
concentration (ng/µl)	325,04	561,15

Mini-Prep and test digestion of the CD biobricks

Investigator: Kira

Ohne Worte *lach*

Wh did you digest with eco and ndeI?? is there a spechía reasonn for it?? if the coonstruct was in the rfc standard could digest it with the igem standard enzymes??

components	sample /µl
DNA	2,5
BSA (100x)	0
Buffer ___4_ (10x)	1,5
Enzyme NdeI (no.Lab:___)	0,5
Enzyme EcoRI (no.Lab:___)	0,5
H₂O	10
Total volume (e.g. 15,20,25,30 µl)	\|15

Incubation: 1 h

Sample 1 was sent for sequencing

109. labday 03.09.2010

Miniprep and test digestion of pSB1C3_lITR_pTERT_ßglobin_mVenus_hGH_rITR

Investigator: Achim

Comment: The test digestion did not show the expected fragments of 2000 and 2500 bp. the photo was exposed too long to see distinct bands. Digestion will be repeated.

Sequencing results

Investigator: Hanna

Comment: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker were cloned and sent for sequencing yesterday.

1. pCerulean_Zegfr:1907: Sequencing looked well.

2. pCerulean_6xHis_MiddleLinker: Sequencing looked also well.

>Cloning of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

Investigator: Hanna

Comment: For N-terminal fusion to VP2, the Affibody - fused to different kinds of linkers - will be cloned into the expression plasmid pCerulean. For imaging CFP, which was fused to the middle linker, will be cloned into pCerulean.

Practical Cloning:

plasmid:
- Vector: name: pCerulean number: P273
- Insert: name: pSB1C3_Zegfr:1907_LongLinker (P298), pSB1C3_Zegfr:1907_MiddleLinker (P290), pSB1C3_Zegfr:1907_SEG (P296), pSB1C3_Zegfr:1907_ShortLinker (P292), pSB1C3_CFP_MiddleLinker (P276)
new vector name: pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker
buffer used: 4 ; Restriction-enzymes used: Enzyme 1 EcoRI-HF ; Enzyme 2 PstI-HF
DNA concentration (vector): 419.5 ng/µL ; DNA concentration (insert): P298 229.7 ng/µL, P290 227.4 ng/µl; P296 218.7 ng/µL; P292 196.6 ng/µL; P276 207.7 ng/µL

Comments: No.

Digestion

components	volume of pCerulean /µl	volume of Zegfr:1907_MiddleLinker /µl	volume of CFP_MiddleLinker /µL
DNA	8.3	7.6	4.8
BSA (10x)	-	-	-
Buffer 4 (10x)	2	2	2
Enzyme 1 EcoRI-HF	1	1	1
Enzyme 2 PstI-HF	1	1	1
H₂O	7.7	8.4	11.2
Total volume	20	20	20

Incubation: 1.5 h

Agarose-Gel:

0.8 g Agarose, 50 TAE (1.5 %), 5 µL EthBr, at 70 Volt, running time: ~ 1 hour

Sample	Sample/µl]	Loading dye (6x)/µl	Expected size 1
P273	20 µl	4 µl	3922 bp
P298	20 µl	4 µl	273 bp
P290	20 µl	4 µl	261 bp
P296	20 µl	4 µl	345 bp
P292	20 µl	4 µl	249 bp
P276	20 µl	4 µl	801 bp

Marker: GeneRuler ladder mix

	Marker	Sample P273 /µl	Sample P276 /µl	Sample P290 /µl	Sample P292 /µl	Sample P296 /µl	Sample P298 /µl
Lane	5	24	24	24	24	24	24

Gel extraction

Gel measurement:

Sample	Volume	Concentration
P273	20	115.45 ng/µL
P276	20	9.91 ng/µL
P298	20	5.07 ng/µL
P296	20	9.03 ng/µL
P290	20	10.31 ng/µL
P292	20	20.78 ng/µL

Ligation

1. PCerulean_Zegfr:1907_LongLinker

	P298	P273
Volume/µl	6.61	1.39

2. PCerulean_Zegfr:1907_MiddleLinker

	P290	P273
Volume/µl	5.53	2.47

3. PCerulean_Zegfr:1907_SEG

	P296	P273
Volume/µl	6.17	1.83

4. PCerulean_Zegfr:1907_ShortLinker

	P292	P273
Volume/µl	4.11	3.89

5. PCerulean_CFP_MiddleLinker

	P276	P273
Volume/µl	7.02	0.98

Trafo

Trafo was performed following the standard protocol. Used cells: XL1b, DNA amount: 2.5 µL of each ligation reaction. Plates were stored @ 37°C room over night.

Harvest viral particles, Seeding HT1080 cells

Investigator: Adrian, Kerstin

Harvest viral particles of Transfection from 31.08.2010 (six approaches: 10µg of RC (2x P326 and 2x P325 and 2x Stratagene), pHelper and GOI (P262))
Seeding HT1080 cells

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

Investigator: Patrick

The Miniprep was performed according to the standard protocol. Labelling:

P381: pCerulean_middlelinker clone 1 upper band : 526,0 ng/µl
P382: pCerulean_middlelinker clone 2 upper band : 522,0 ng/µl
P383: pCerulean_middlelinker clone 3 upper band : 463,0 ng/µl
P384: pCerulean_middlelinker clone 1 lower band : 465,0 ng/µl
P385: pCerulean_middlelinker clone 2 lower band : 500,0 ng/µl
P386: pCerulean_middlelinker clone 3 lower band : 524,0 ng/µl

Öhm Patrick: Hier ist der Grund, warum das Experiment "wiederholt" wurde: Falsche Beschrieftung hier, als auch in der Excel Tabelle ;) "Finde den Fehler!" :P

;-) bitte ausfüllen

see also: http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Cloning_CFP_middlelinker_.28from_pSB1C3_CFP_middlelinker.2C_P276.29_into_pCerulean_.28P273.29
Testdigestion: 7 µl DNA sample, 1 µl Buffer 4 (10x), 1 µl BSA, 0,5 µl Xba, 0,5 µl PstI-HF
Expected size of the fragments: 786 & 2053bp
There seems to be something wrong.

Sent for sequencing: P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev
Used primer: O21 : CMV_reverse_qPCR

Cloning of pSB1C3_leftITR_pTert and pSB1C3_mGMK with pSB1C3_leftITR_CMV

Investigator: Chris L.

Vector: name: pSB1C3_leftITR_pTERT clone 1 P256 c=179,2 ng/µl
Vector: name: pSB1C3_leftITR_CMV P188 c=231,6 ng/µl
Insert: name: pSB1C3_mGMK P195 c=258,6 ng/µl
new vector name: pSB1C3_leftITR_CMV_GMK
new vector name: pSB1C3_leftITR_pTERT_GMK
buffer used: 4 ; Restriction-enzymes used: Enzyme 1 PstI ; Enzyme 2 SpeI ; Enzyme 3 XbaI

components	P195	P256 /µl	P188 /µl
DNA	7,7	11,2	8,6
BSA (10x)	2	2	2
Buffer 4 (10x)	2	2	2
Enzyme SpeI	1	1	0
Enzyme XbaI	0	0	1
Enzyme PstI	1	1	1
H2O	6,3	2,8	5,4
Total volume (e.g. 15,20,25,30 µl)	20	20	20

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45

Results:

c(Insert)= 15,37 ng/µl; size: 627 bp
c(Vector)= 50,84 ng/µl; size: 2869 bp
c(Vector)= 75,64 ng/µl; size: 2672 bp

Ligation of PCR products and vector:

For the Ligation 1µl T4 buffer (10x) and 1µl T4 ligase were used. Incubation time: 45 min

	pSB1C3_leftITR_CMV + GMK /µl	pSB1C3_leftITR_pTERT + GMK
Vector	5,48	6,21
Insert	2,52	1,79

Transformation:

The transformation was done following the standard protocol using XL1 blue cells.

Sequence analysis of pCerulean_VP1up

Investigator: Bea

The assembled pCerulean_VP1up which serves as the first construct in the VP1 insertion cloning assembly was sent for sequencing.

Plasmid used: P365
Primer used: CMV-F
Tube name: SB1
Folder (Geneious): N-terminal Targeting --> pCerulean_VP1up_NLS

Freiburg 10 SeqAnalysis of pCerulean VP1up NLS 03.09.2010.jpg

pCerulean_VP1up_NLS_Targeting molecule

Investigator: Bea

Comment: For preparing the next step in the VP1 insertion, different targeting molecules will be fused to the construct pCerulean_VP1up_NLS.

The first construct is the Affibody Z_EGF-R:1907 which was already cloned into the standard iGEM plasmid and can be sued for target tumor cells overexpressing the EGF receptor.
The second construct is the mVenus (Yellow fluorescent protein) which can be used for imaging experiments
Another motif will be the His-tag. Since the His-tag is very small, the oligos will be hybridized and directly ligated into the pCerulean backbone.

Digestion of vector:

Plasmid used: pCerulean_VP1up_NLS (P365) c=470 ng/µL
Add BSA
AgeI-HF and SpeI-HF were used

Protocol of the digestion of the vector:

Components	v_{pCerulean_VP1up_NLS}/µL
DNA	3
BSA (100x)	2,5
Buffer no. 4 (10x)	2,5
Enzyme 1 AgeI	1
Enzyme 2 SpeI HF	1
H₂O	15
Total volume	25

Incubation of the sample at 37°C
Incubation for 120 minutes

The Targeting molecules were digested with different enzmyes than the vector for providing the ability to assemble the different constructs together without creating new restriction site but to delete them. This works by fusing NgoMIv and AgeI together.

Digestion of the targeting molecules

Plasmids used:

pSB1C3_zEGF-R:1907 (P284) c=146 ng/µL
pGA14_mVenus(P60) c=280 ng/µL

Add BSA
NgoMIV and SpeI-HF were used

Components	v_{ZEGFR (P284)}/µL	v_{pGA_mVenus (P60)}/µL
DNA	13 µl	7 µl
BSA (10x)	2,5 µl	2,5 µl
Buffer no. 4 (10x)	2,5 µl	2,5 µl
Enzyme 1 NgoMIV-	1,0 µl	1,0 µl
Enzyme 2 AgeI HF	1,0 µl	1,0 µl
H₂O	15 µl	15 µl
Total volume	25	25

Incubation of the sample at 37°C
Incubation for 90 minutes

After incubation the samples were loaded on a 1% agarose gel. The obtained gel (after running the gel for 15 minutes) can be seen below.

Freiburg10 pCerulean VP1up NLS TargetingMolecule.jpg

Results: The boxes mark fragments which ere cut out of the gel. The left lane (pCerulean) looks a bit strange, because Marker was added after the digestion reaction instead of Loading dye.

Parallel, for assembling the His-Tag to the construct pCerulean_VP1up_NLS, oligos for the His-Tag in the RFC25 standard (provided by Gerrit) were hybridized and can directly be ligated in the digested vector. This strategy was used because of the smal size of the His-Tag which cannot be separated easily by the agarose gel.

Hybridization of the His-Oligos

10µL oligo1 (1:10)
10µL oligo2 (1:10)
4µL 100mM Tris-Cl pH 8
10µL 5mM MgCl₂
8µL H₂O

The used programm for the hybridization:

99°C 7´
99°C 1´
-1°C R=0,3°/s
Goto 2 rep 74
Hold 4°C

After the gel extraction of a T4 ligation was performed.

Affibody Z_EGF-R:1907:

v_{Cerulean_VP1up_NLS} = 5,82µL
v_ZEGF-R:1907 = 2,18µL

mVenus Z:

v_{Cerulean_VP1up_NLS} = 4,04µL
v_mVenus = 3,96 µL

6xHisTag:

v_{Cerulean_VP1up_NLS} = 0,2µL
v_6xHisTag = 7,98µL

Next steps: Picking clones of the trafo plates (containing Kanamycin) and perform the Mini-Prep. The obtaining constructs finally will be fused to the VP2/3 protein which results in the final construct for the VP1 targeting approach.

3rd Repetition of Mini-Preps and test digestion for Loop insertion BioBricks

Investigator: Achim, Anna

Sequencing results of Loop insertion BioBricks from 02.09.10; the following clones looked well:

pSB1C3_587_KO_RGD_clone6.4
PSB1C3_587_KO_His_clone9.4

Update: 9 of the 14 ViralBricks looked well (sequencing results from 01. - 03.09.). Two new clones of the remaining constructs were prepared and test digested.

Test digestion:

components	Volume for each sample /µl
DNA	10
BSA (10x)	1,5
Buffer 4 (10x)	1,5
Enzyme EcoRI HF	0,5
Enzyme NotI HF	0,5
H2O	1
Total volume /µl	15

Components	453 BAP	453 Z34C	587 Z34C	587 KO Z34C	587 KO Z34C SPACER
Clone	1.5 + 1.6	11.5 + 11.6	12.5 + 12.6	13.5 + 13.6	14.5 + 14.6

Incubation time: 1 h, Incubation temperature: 37°

Preparation of gel:
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

Expected fragment sizes /bp:
pSB1C3: 2051 bp
BAP: ~150
Z34C: ~ 200

Comment: The following clones were sent for sequencing: 1.6, 12.6 ,13.6 and 14.5.
To do: Retrafo of clone6.4 and clone9.4 and preparation of glycerol stocks.

Cloning of Cap into pAAV_RC_ins-rep: preparation for SDM

Investigator: Stefan

Comment: Cloning Cap into pAAV still does not work as planned. As another approach Cap can be cut using BsiWI and XcmI and performing a SDM in the part of Cap not cloned into pAAV. Primers are ordered and should arrive on monday to proceed.

Digestion:

components	pMA_RepCap Vector_SDM_InsPvuII (P211)	pAAV_RC_ins-rep (P250)
DNA	7,9	2,1
Buffer 2 (10x)	2	2
XcmI	1	1
BsiWI	1	1
H₂O	8,1	13,9
Total volume	20	20

Comment:Digestion was performed using two steps, first incubating for 1 hour at 37 °C, afterwards for 1,5 hours at 55 °C.

Gel:

0,5 g Agarose,50 ml TAE (1%), 5 µl EtBr , at 115 Volt, running time: 50 minutes

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

pMA_RepCap Vector_SDM_InsPvuII (P211): c= 6,26 ng/µl
pAAV_RC_ins-rep (P250): c= 13,72 ng/µl

Quick Ligation:

Comment: XcmI produces only 1 base overhang, therefore Quickligase was used for ligation and incubation time increased.

The Ligation was performed as following:

Vector Volume: 4,59 µl
Insert Volume: 4,41 µl

10 µl QuickLigase buffer (2x)
9 µl (Vector + Insert) mix
1 µl QuickLigase

Incubating for 60 minutes.

Transformation:

Trafo was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

110. labday 04.09.2010

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273): Sequencing results of P381 and P385

Investigator:Patrick

Sequencing results of P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev: A wrong primer was used so there are no relevant sequence data yet. On sunday i will sent these clones again for sequencing. Primer: GATC_std_CMV-F

Ha ich war schneller als Bea beim "Kopfkratzen-setzen" *lol*

Hiermit verspreche ich dass es noch jede Menge Kopfkratzer, Laboraufsichtswichtel und pinke Panter geben wird (Patrick)

Harvest viral particles, Transduction

Investigator:Kerstin, Adrian

Harvest viral paricles from one plate of transfection from 01.09.2010 (10 plates, same treatment (10µg of each plasmid P357, P263, P356))
Transduction:

1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P326)
1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P325)
4x 6-well: 10x 0,5ml and 4x 1ml of virus from 01.09.2010 (stratagene R/C)
4x 6-well: 10x 0,5ml and 4x 1ml of virus from 04.09.2010 (10 plates same treatment)
6x 6-well: 0,5ml of virus from 21.08.2010 (TKGMK; 18x pH 7,10 and 6x pH 7,12)

Repetition of Hybridisation and Cloning for Loop insertion BioBricks

Investigator: Achim, Anna

Samples:

1: 453_BAP
11: 453_Z34C
12: 587_Z34C
13: 587_KO_Z34C
14: 587_KO_Z34C_Spacer

Comment: New approaches of the Hybridisation and Fill-in reactions were done because it didn't work the first time. A possible reason is that the klenow enzyme wasn't inactivated. In addition, the conditions for ligation will be improved. Also the ligation of 453_BAP was done again.

Digestion:

components	Volume Vector 587	Volume Vector 453	Sample 11	Sample 12	Sample 13	Sample 14
DNA	3,7	3,7	6	6	6	6
BSA (10x)	-	-	-	-	-	-
Buffer 4 (10x)	2	2	2	2	2	2
Enzyme 1°	Bam	Ssp	Ssp	Bam	Bam	Bam
Enzyme 2°	Pvu	Sal	Sal	Pvu	Pvu	Pvu
H2O	12,3	12,3	10	10	10	10
Total volume /µl	20

° 1 µl each

Preparation of gel:
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 50 minutes

Repetition: Prepatation for SDM: cloning of Cap into pAAV

Investigator: Stefan

Schönen Abend dir noch

Comment: On yesterday's plates nothing grew. Because gelex and ligation products were stored in coldroom, new ligation and trafo approaches were performed.

Ligation approaches:

ligation with Quick Ligase (as performed yesterday)
ligation with T4 Ligase

Transformation approaches:

Quick Ligase (approach from yesterday):
- 2 µl of ligation product
- 4 µl of ligation product

Quick Ligase (new approach):
- 2 µl of ligation product
- 4 µl of ligation product

T4 Ligase:
- 2 µl of ligation product
- 4 µl of ligation product

Transformation:

Trafo of each approach was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

Ligation and trafo of the modified Cap insert , the Viral Bricks 1, 11, 12, 13 and 14

Investigator: Volker

Ligation

Construct	Vector (µl)	Insert(µl)
ViralBrick 1 in 453	7.91	0.09
ViralBrick 11 in 453	6.17	1.83
ViralBrick 12 in 587	6.15	1.85
ViralBrick 13 in 587	5.3	2.7
ViralBrick 14 in 587	4.46	3.54
p211 in pAAV-RC	5.52	3.54
p211 in pAAV-RC	5.52	3.54

Preparation of PBS Buffer

Investigator: Volker

Two liters of PBS buffer were prepared for the column purification of the viral particles. The following protocol was used:

dissolve the following in 800ml ddH₂O:
- 8g of NaCl
- 0.2g of KCl
- 1.44g of Na₂HP0_$ (1.69 because of the crystal water in the reagent)
- 0.24 g of KH₂PO₄
adjust to pH 7.4
adjust to 1000ml
steril filtrate the solution

111. labday 05.09.2010

Mini-prep of glycerol stock pMA_RepCap Vector_SDM_InsPvuII clone 1 (B182)

Investigator: Stefan

Glycerol stock: Because glycerol stock B182 was not mixed before putting into -80 °C freezer the cells died. Therefore a new glycerol stock was prepared and labled B302.

Mini-Prep was performed according to the standard protocol. Two preps were prepared:

P363 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (1) = 215,6 ng/µl
P364 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (2) = 230,5 ng/µl

>Picking clones of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

Investigator: Hanna

2 clones of each construct (1. pCerulean_Zegfr:1907_ShortLinker, 2. pCerulean_Zegfr:1907_MiddleLinker, 3. pCerulean_Zegfr:1907_LongLinker, 4. pCerulean_Zegfr:1907_SEG and 5. pCerulean_CFP_MiddleLinker) were picked.

To do tomorrow (6.9.): Mini-Preps and Test digestion (with EcoRI and PstI).

112. labday 06.09.2010

Minipreps & Test PCR of pTert Biobrick assembly

Investigator: Achim

After unsucessful test digestion with EcoRI and PstI I tried out a test PCR with Igem Primers (same Mastermix as for loop inserts)
Elongation time was set to 2.35 min because the amplified fragment should be about 2.5 kbp big.
Result:

The visible bands are not specific. Either cloning or PCR went wrong :)

Next steps: Another test digestion tomorrow, if that doesn't work out I'll repeat the ligation.

Minipreps & Test PCR of Loop inserts 1, 11, 12 ,13, 14

Investigator: Achim & Anna

Samples:

1: 453_BAP
11: 453_Z34C
12: 587_Z34C
13: 587_KO_Z34C
14: 587_KO_Z34C_Spacer

Minipreps of 1.1, 1.3, 11.1, 11.2, 12.1, 12.2, 13.1, 13.2, 14.1, 14.2
Clones 1.4, 11.3, 11.4, 12.3, 12.4, 13.3, 13.4, 14.3, 14.4 were pelleted and stored away in case the first two are incorrect.

In the hope to confirm our small inserts, A test PCR using Igem standard primers was carried out:

PCR-reaction:

Volume / µl	ingredients
2,5	Taq Buffer
x	template (~0.5 ng)
2,5	forward Igem primer (1:10)
2,5	reverse Igem primer (1:10)
0,5	dNTP
filled upt to 25	H₂O
0,5	Taq Polymerase (1.25)

PCR program:

Rounds	temperature/ °C	Time
1	95	30"
	95	25"
25	61	30"
	68	50"
1	68	5"

Results:

Bands should be about 150 for BAP and about 200 for Z34C. Bands dont quite match the expectations, somehow the BAP band seems bigger than the Z34C band. Clones 1.3, 11.1, 12.2, 14.1 were sent for sequencing.

Mini-Preps and test digestion of Linker BioBricks

Investigator: Anna

Comment: Mini-preps of all clones were prepared. They have not been added to the excel sheets yet, but were stored in the freezer -> Samples of the usable and sequenced constructs were added to the excel sheets and stored in the freezer (06.09.10).

Samples	1	2	3	4	5	6
Insert	VP1up_NLS_His	VP1up_NLS_His	ZeGFP:1907_SEG	ZeGFP:1907_SEG	ZeGFP:1907_ML	ZeGFP:1907_ML
Clone	1	2	1	2	1	2
Concentration	439,57	463,51	415,24	354,5	406,75	382,27

7	8	9	10	11	12
CFP_ML	CFP_ML	ZeGFP:1907_SL	ZeGFP:1907_SL	ZeGFP:1907_LL	ZeGFP:1907_LL
1	2	1	2	1	2
482,76	424,76	497,06	413,55	391,05	427,20

SL: Shortlinker ML: Middlelinker LL: Longlinker

Test digestion:

components	Volume for each sample /µl
DNA	1
BSA (10x)	-
Buffer 4 (10x)	1
Enzyme EcoRI HF	0,5
Enzyme Pst HF	0,5
H2O	7
Total volume /µl	10

Preparation of gel:

Incubation time: 1 h, Incubation temperature: 37°
1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

Expected fragment sizes:

VP1up_NLS_Histag: 550 bp
ZeGFP:1907_SEG: 344 bp
ZeGFP:1907_ML: 261 bp
CFP_ML: ~ 1000 bp
ZeGFP:1907_SL: 249 bp
ZeGFP:1907_LL: 237 bp

To do: Test digestion of VP1up_NLS_mVenus and Affibody. Number 2, 3, 5, 7, 9 and 12 will be sent for sequencing tomorrow. For sequencing results of VP1up_NLS_Histag go to labday 09.09.2010, for the linker BioBricks go to labday 08.09.2010.

Production of Ampicilin and Chloramphenicol

Investigator: Jessica

10ml ethanol (70%) containing 1g Amp in 60µl aliquots
10ml ethanol (70%) containing 0,25g Cm in 60 µl aliquots

Mini-prep and test digestion of pAAV_RC-CapIns_prepSDM

Investigator: Stefan

Mini-Prep was performed according to the standard protocol

P394 = pAAV_RC_CapIns_prepSDM clone 1 = 296,42 ng/µl
P395 = pAAV_RC_CapIns_prepSDM clone 2 = 274,92 ng/µl
P396 = pAAV_RC_CapIns_prepSDM clone 3 = 498,39 ng/µl
P397 = pAAV_RC_CapIns_prepSDM clone 4 = 487,67 ng/µl
P398 = pAAV_RC_CapIns_prepSDM clone 5 = 469,96 ng/µl
P399 = pAAV_RC_CapIns_prepSDM clone 6 = 517,06 ng/µl
P400 = pAAV_RC_CapIns_prepSDM clone 7 = 451,68 ng/µl
P401 = pAAV_RC_CapIns_prepSDM clone 8 = 479,18 ng/µl

Constructs were digested with XcmI and Acc65I for 70 minutes at 37°C

components	pAAV_RC_CapIns_prepSDM clone 1-8
DNA	2
Buffer 2 (10x)	1
BSA (10x)	1
XcmI	0,5
Acc65I	0,5
H₂O	5
Total volume	10

Sent for sequencing: Clone 3 (P396) was sent for sequencing.

Tube name: SB1
Primer used: VP 4200 rev

Sent for sequencing

Investigator: Stefan

Tube name: SB2

pAAV_RC_inserts clone1 (P326) was sent for sequencing using the following primers:

VP1 primer for pKex rev
SK
Primer Cap 2800 for
Primer Cap 2800 rev
Primer Cap 3500 for

113. labday 07.09.2010

Sequencing results of new loop insertion ligations

Investigator: Achim

Sequencing confirmed the 453 BAP ligation contains the correct sequence. 453 Z34C and 587 Z34C didn't contain inserts, 587 KO Z34C still contained the BLA placeholder.

Repetition of CD biobrick production

Investigator: Kira
Comment: Despite the results of the test digestion, the samples, which were sent for sequencing do not contain the GOI. Thus the whole procedure will be repeated today.

DNA sample was diluted 1:100

Ingredients	CD sample
5X Phusion HF buffer	10 µl
10 mM dNTP mix	1µl
forward primer: O158	2,5µl
reverse primer: O159	2,5 µl
DNA Template	0,5 µl
DMSO	0 µl
Phusion Polymerase	0,5 µl
H₂O	33µl
Total volume	50 µl

PCR program:

Cycles	Temperature	Time
	98°C	1
8x	98°C	15"
	60°C	25"
	72°C	40"
17x	98°C	15"
	65°C	25"
	72°C	40"
1x	72°C	5'
Hold 4°C

Digestion of plasmid backbone:

c (pSB1C3) = 151, 1 ng/ µl

Components	vector Volume/µL
DNA 1 µg	6,0 µl
BSA (100x)	0,2 µl
Buffer no. 4 (10x)	2,0 µl
Enzyme 1 XbaI	0,5 µl
Enzyme 2 AgeI HF	0,5 µl
H₂O	10,8 µl
Total volume	20

incubation @ 37 C for approx. 2 h

1% agarose gel

Digestion of PCR product:

Components	PCR product Volume/µL
DNA	30,0 µl
BSA (100x)	0,4 µl
Buffer no. 4 (10x)	4,0 µl
Enzyme 1 XbaI	1,5 µl
Enzyme 2 AgeI HF	1,0 µl
H₂O	3,1 µl
Total volume	40

incubation @ 37 C for approx. 2 h

Ligation

Quickligase was used

DNA-mix 9 ul (8,2 ul insert +0,8 ul insert)
Quickligase 1ul
Quickligase Buffer 10 ul

Incubation @ RT for 10'

Transformation

BL21 cells were used for transformation because XL1B cells have to be tested first. Transformation according to the standard protocol.

Comment: to figure out if, the digestion of the vector and the following ligation was successful, 2 plates have been prepared. One plate contains only the digestion vector, while the second contains the ligated sample.

Test digestion of pCerulean_VP1up_NLS_mVenus and pCerulean_VP1up_NLS_Affibody

Investigator: Anna

Samples	Insert	Clone	Concentration [ng/µl]
1	VP1up_NLS_mVenus	1	416,59
2	VP1up_NLS_mVenus	2	387,46
3	VP1up_NLS_Affibody	1	390,68
4	VP1up_NLS_Affibody	2	374,12

Test digestion:

components	Volume for each sample /µl
DNA	2
BSA (10x)	-
Buffer 4 (10x)	1
Enzyme EcoRI HF	0,5
Enzyme Pst HF	0,5
H2O	6
Total volume /µl	10

Preparation of gel:

Incubation time: 2 h, Incubation temperature: 37°
0,5 g Agarose, 50 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

Expected fragment sizes:

VP1up_NLS_mVenus: 1215 bp
VP1up_NLS_Affibody: ~ 700 bp

Comment: Samples 1 and 3 were sent for sequencing. For sequencing results go to labday 09.09.2010.

Reftrafo of pSB1A2_EGFP_HQ from distribution plate (Bba_I719015 plate 1 well 15P = T7GFPmut3bHQ)

Investigator: Jessica
P15 (T7GFPmut3bHQ) from plate 1 of the distribution plates from the iGEM headquarter was solved in 10 µl H2O

two trafos was prepared with 2 µl DNA and XL1blue (lab)
- plate 1: 90µl DYT + 10µl mixed DNA-XL1b-solution
- plate 2: resupended pellet

Design and Ordering of GFP-Oligos

Investigator: Hanna

Comment: In order to perform single virus tracing experiments and in order to test the Affibody and the DARPin, we decided to use GFP which can be better visualized than CFP. Because we received GFP in the distribution kit we performed a trafo with the Bba_I719015. In addition to that we want to convert the GFP into the RFC25 standard - because for our purpose we want to be able to perform fusion approaches.

Sequence analysis of pAAV_RC_CapIns_prepSDM

Investigator: Stefan

The synthetised Cap was inserted into the pAAV vector already containing the 4 mutations and the Rep construct.

Tube name: SB1
Primer used: VP 4200 rev

Freiburg10 Sequencing pAAV RC CapIns prepSDM.jpg

Cloning of Cap into pAAV_RC_1.2 SDM SalI

Investigator: Stefan

Comment: pAAV with inserted Rep does not work in cell culture as expected. It was assumed that this is related to the deletion of a restriction site for KpnI at the splicing site of Rep40/Rep68. To test our synthethised Cap a plasmid only containing this will be produced.

Digestion:

components	pMA_RepCap Vector_SDM_InsPvuII (P392)	pAAV_RC_1.2 SDM SalI (P158)
DNA	11,6	3,7
Buffer 2 (10x)	2	2
XcmI	1	1
BsiWI	1	1
H₂O	4,4	12,3
Total volume	20	20

Comment:Digestion was performed using two steps, first incubating for 1 hour at 37 °C, afterwards for 1,5 hours at 55 °C.

Gel:

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 50 minutes

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

pMA_RepCap Vector_SDM_InsPvuII (P392): c= 8,43 ng/µl
pAAV_RC_1.2 SDM SalI (P158) c= 17,24 ng/µl

T4 Ligation:

Comment: XcmI produces only 1 base overhang, but using Quick Ligase is not working properly. Therefore ligation is performed using T4.

The Ligation was performed as following:

Vector Volume: 4,17 µl
Insert Volume: 3,83 µl

1 µl T4 ligase buffer (10x)
8 µl (Vector + Insert) mix
1 µl T4 Ligase

Incubating for 70 minutes.

Transformation:

Trafo was performed according to the standard protocol (BL21). The cells were plated on a agar plate with ampicilin.

Repetition of Mini-Preps and test digestion of Linker BioBricks from 2nd ligation

Investigator: Achim, Anna

Comment: Recent update of succesfully produced ViralBricks:

File:Freiburg10 Production of ViralBricks.pdf

Test digestion:

Number	11.4	11.4	12.3	12.4	13.3	13.4	14.3	14.4
DNA	7	6,5	5,5	5	5	12,52	8,5	7
H2O	5,5	6	7	7,5	7,5	0	4	5,5

BSA: no
Enzymes:
EcoR I HF: 0,5 µl
Pst I HF: 0,5 µl
Total volume /µl:10

Incubation time: 3/4 h, Incubation temperature: 37°

Preparation of gel:

1 g Agarose, 50 ml TAE (2%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

To do: A new approach of the ViralBricks production has to be done tomorrow. A possible reason for the failure of the reactions is that the restriction enzymes didn't cut in the klenow buffer.

Preparation for Midi-Preps of pAAV_mVenus, pHelper and pAAV_RC

Investigator: Kerstin

25 ml DYT was prepared with 25 µl Ampicillin and inoculated with glycerol stocks of pAAV_mVenus, pHelper and RC, each were incubated over night in 37°C room.

Sequencing

Investigator: Hanna
pCerulean_Zegfr:1907_ShortLinker, pCerulean_Zegfr:1907_MiddleLinker, pCerulean_Zegfr:1907_LongLinker, pCerulean_Zegfr:1907_SEG and pCerulean_CFP_MiddleLinker were sent for sequencing.
Used primer: GATC_std_CMV-F

114. labday 08.09.2010

Sequencing results of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

Investigator: Hanna

Comment: In order to perform single virus tracing experiments, CFP, which was fused to the Middle-Linker, was cloned into pCerulean. Referring to the establishment of a small linker library, the Affibody Zegfr:1907 was fused to the Short-Linker, Middle-Linker, Long-Linker and SEG-Linker and was then cloned into pCerulean.

Sequencing results:
1. pCerulean_CFP_MiddleLinker:

Conclusion: Sequencing looked well, CFP_MiddleLinker was cloned successfully into pCerulean is now ready for fusion to VP2/3.

2. pCerulean_Zegfr:1907_ShortLinker:

Conclusion: Sequencing looked also well, Zegfr:1907_ShortLinker was cloned successfully into pCerulean is now ready for fusion to VP2/3.

3. pCerulean_Zegfr:1907_MiddleLinker:

Conclusion: Sequencing looked also well, Zegfr:1907_MiddleLinker was cloned successfully into pCerulean is now ready for fusion to VP2/3.

4. pCerulean_Zegfr:1907_LongLinker:

Conclusion: Sequencing delivered that there was a C -> G transistion between the NgoMIV restriction site and the Zegfr:1907 sequence. Because it's located in the gene-synthesis region, the question is, whether there was a mutation caused by the XL1b cells or whether sequencing quality is not good enough. Therefore it was decided to sequence this conbstruct once again in reverse direction.

5. pCerulean_Zegfr:1907_SEG:

Conclusion: Sequencing looked also well, Zegfr:1907_SEG was cloned successfully into pCerulean is now ready for fusion to VP2/3.

Repetition of PCR of SV40 terminator

Investigator: Anna

Comment: Second repetition of SV40 PCR, insert and vector were cut with Age I and Xba I.

PCR sample: p230 (PeGFP_C1)
Plasmid backbone: p51.2 (pSB1C3_CFP)

Ingredients	CD sample
5X Phusion HF buffer	10 µl
10 mM dNTP mix	1µl
forward primer: O158	2,5 µl
reverse primer: O159	2,5 µl
DNA Template	3 µl
DMSO	0 µl
Phusion Polymerase	0,5 µl
H₂O	30,5 µl
Total volume	50 µl

PCR program:

Cycles	Temperature	Time
	98°C	1
8x	98°C	15"
	59°C	25"
	72°C	7"
17x	98°C	15"
	67°C	25"
	72°C	7"
1x	72°C	5'
Hold 4°C

Digestion of plasmid backbone:

c (pSB1C3_CFP) = 151, 1 ng/ µl

Components	vector Volume/µL
DNA 1 µg	7,0 µl
BSA (100x)	2,0 µl
Buffer no. 4 (10x)	2,0 µl
Enzyme 1 XbaI	0,5 µl
Enzyme 2 AgeI HF	0,5 µl
H₂O	8 µl
Total volume	20

incubation @ 37 °C for approx. 2,5 h

Preparation of gel:

0,75 g Agarose, 50 ml TAE (1,5%), 6 µl GELRED , at 115 Volt, running time: 45 minutes
Expected size of fragment: 261 bp

Digestion of PCR product:

Components	PCR product Volume/µL
DNA	18,5 µl
BSA (100x)	3 µl
Buffer no. 4 (10x)	3 µl
Enzyme 1 XbaI	1 µl
Enzyme 2 AgeI HF	1 µl
H₂O	3,5 µl
Total volume	30

incubation @ 37 °C for approx. 2 h

Ligation

c(pSB1C3_cut_Age_Xba) = 0,9 ng/µl
c(Insert) = 25,8 ng/µl

T4 Ligase was used.

DNA-mix 8 ul (7,08 µl insert + 0,92 µl insert)
T4 Ligase 1µl
T4 Ligase Buffer 1 µl

Incubation @ RT for 20'

Transformation

Transformation was done according to the standard protocol with XL1B cells.

PCR of P40

Investigator: Stefan
DNA sample was diluted 1:1000

PCR progam:

Ingredients	Volume / µl
5x Phusion HF buffer	10
10 mM dNTP mix	1
forward primer: O152	2,5
reverse primer: O153	2,5
DNA Template	1
DMSO	-
Phusion Polymerase	0,5
H₂O	31,5
Total volume	50

PCR program:

Cycles	Temperature / °C	Time / s
	98	60
8x (step 2 - 4)	98	15
	61	25
	72	3
17x (step 5- 7	98	15
	69	25
	72	4
1x	72	300
Hold	4

Digestion of plasmid backbone:
Plasmid used: pSB1C3_CFP (P51.2)
c (pSB1C3_CFP) = 151, 1 ng/ µl

Components	vector Volume/µL
DNA	10
BSA (10x)	2
Buffer no. 4 (10x)	2
XbaI	1
PstI	1
H₂O	4
Total volume	20

incubation @ 37 C for approx. 2 h

Comment: PCR does not work out twice. Therefore troubleshouting will have to be done.

Midi-Prep

Investigator: Anissa, Kerstin

Midi-Prep was performed with B34 (pAAV_iGEM_mVenus_YFP), R/C (standard P357) and pHelper (standard P356) according to the standard protocol.

c(pHelper): 518,7 ng/µl
c(RC): 1068,7 ng/µl
c(mVenus): 277,9 ng/µl

Seeding HT1080, Passaging AAV293 and Transfection

Investigator: Kerstin, Patrick

Seeding HT1080 cells for Transduction at 09.09.2010: two 6-well plates were prepared (200.000 cells per well)
Passaging AAV293 cells from T75 to T175 flasks
Transfection of AAV293: Ten plates same treatment (10µg of each plasmid):
- c(RC)= 1068,7 ng/µl --> 9,4 µl
- c(pHelper)= 518,7 ng/µl --> 19,3 µl
- c(TKGMK, P81b)= 1900 ng/µl --> 5,3 µl

Checking fractions of Äkta run

Investigator: Stefan

Comment: Fractions 6 - 11 of supernatant and of lysated cells treated with benzonase have to be checked for virus particles as well as fraction 20 of the second approach. This will be done amplifing the CMV promotor. Additionally, one positive control (pCerulean P274) and one negative control (HT1080 cells already confirmed being negative in qPCR) were used for PCR approach.

PCR progam: The following primers were used:

CMV_forward_qPCR
CMV_reverse_qPCR

Ingredients	Volume / µl
10x ThermoPol buffer	10
10 mM dNTP mix	1
forward primer: O20	2,5
reverse primer: O21	2,5
MgCl₂ (50mM)	4
fraction	5
DeepVent	0,5
H₂O	8
Total volume	25

PCR program:

Cycles	Temperature / °C	Time / s
	95	600
35x (step 2-4)	95	20
	56	20
	72	18
1x	72	120
Hold	4

Gel run will have to be performed tomorrow.

115. labday 09.09.2010

Picking clones of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks

Investigator: Kira
The plates were checked in the morning and they did not contain any visible colonies, thus the plates were kept @ 37C till the late afternoon in order to check again.

Fortunately, after approx. 18h of incubation the plate observation revealed lots of colonies, thus 3 clones from each plate could be inoculated in the DYT media.

new ligation try and transformation of CD-biobrick

Investigator: Kira

CD-biobrick plate contained just few (around 3 colonies), thus new ligation approach was performed. T4 ligase was used. The ligation mix was incubated @ 18C for around 5 hours

Transformation was performed according to the standard protocol

Repetition of ViralBrick Ligations: 453 Z34C, 587 Z34C, 587 KO Z34C, 587 KO Z34C Spacer

Investigator: Achim

Nomenclature:

11K: 453_Z34C in Klenow Buffer
12K: 587_Z34C in Klenow Buffer
13K: 587_KO_Z34C in Klenow Buffer
14K: 587_KO_Z34C_Spacer in Klenow Buffer

11 4: 453_Z34C in Buffer 4
12 4: 587_Z34C in Buffer 4
13 4: 587_KO_Z34C in Buffer 4
14 4: 587_KO_Z34C_Spacer in Buffer 4

Two new reaction setups for the remaining four ViralBricks:

Hybridisation & Fill-In reaction in Buffer 4, heat inactivation, digestion, ligation
Hybridisation & Fill-In reaction in Klenow Buffer, heat inactivation, PCR Purification, digestion, ligation

We followed the recommendations provided by Fermentas with their Klenow Fragment manual:

2 µl total DNA (3,3 µl per Oligo)
0,1 µl dNTPs
2 µl Klenow Buffer/ Buffer 4

in a 20 µl reaction.

Components	453 Z34C	587 Z34C	587 KO Z34C	587 KO Z34C SPACER
	O143	O145	O147	O149
	O144	O146	O148	O150

Hybridisation was performed using the TKfill program on the gradient cycler. After Hybridisation, 0,5 µl Klenow fragment was added. The fill-in reaction was run for 40 minutes at 37°C. Afterwards, the Klenow fragment was heat inactivated at 75°C for 10 minutes. The samples containing the Klenow buffer were then purified with the QIAquick PCR Purification Kit Protocol.

Samples 11 K and 11 4 were digested with SspI and SalI, Samples 12-14 were digested with BamHI and PvuII. Buffer 4 was added to the purified samples. One sample of the vector plasmid pSB1C3_BLA (P320) was digested with SspI and SalI, two samples were digested with BamHI and PvuII two obtain a sufficient amount of DNA for 6 ligations.

(Table with concentrations and used DNA Volumes)

After digestion, the digested inserts were separated on a 2% agarose gel, the vector backbones were separated on a 1% agarose gel:

(Pictures of gels)

Vektor 587

Vektor 453

Inserts

(Nanodrop concentrations of gelex:)

Ligation:

(Ligation volumes)

Vectors and Inserts were ligated for ~3h with T4 Ligase at room temperature.

Two µl of each ligation were transformed into XL1B cells according to our standard protocol. Plates were incubated overnight

Midiprep of pHelper, pAAV_RC and pAAV_iGEM_mVenus (B34)

Investigator: Patrick
The Midiprep was performed according to the standard protocol yieldung the following concentrations:

pAAV_iGEM_mVenus (B34): 1131,7 ng/µl, labeled P419,420,421
pHelper: 548 ng/µl
pAAV_RC: 985 ng/µl

Miniprep of pAAV_RC_CapIns_noRep and pSB1A2_EGFP_HQ

Investigator: Stefan

Mini-Prep was performed according to the standard protocol

P413 = pAAV_RC_CapIns_noRep clone1 c= 310,73 ng/µl
P414 = pAAV_RC_CapIns_noRep clone2 c= 217,66 ng/µl
P415 = pAAV_RC_CapIns_noRep clone3 c= 427,06 ng/µl
P416 = pSB1A2_EGFP_HQ clone1 c= 252,56 ng/µl
P417 = pSB1A2_EGFP_HQ clone2 c= 255,02 ng/µl
P418 = pSB1A2_EGFP_HQ clone3 c= 227,59 ng/µl

components	pAAV_RC_CapIns_noRep clone1-3	pSB1A2_EGFP_HQ clone1-3
DNA	1	1
Buffer (10x)	1	1
BSA (10x)	1	1
Enzyme 1	Acc65I 0,5	XbaI 0,5
Enzyme 2	BsiWI 0,5	PstI 0,5
H₂O	6	6
Total volume	10	10

Freiburg10 test digestion pAAV CapIns noRep pSB1A2 eGFP HQ.jpg

Sent for sequencing: pAAV_RC_CapIns_noRep clone1 (P413)

Tube name: SB1
Primer used: VP 4200 rev

Sent for sequencing: pSB1A2_EGFP_HQ clone1 (P416)

Tube name: SB2
Primer used: VR2

PCR of VP2/VP3

@jessi: Did you clone the constructs theoretically?? I cannot find it? And is there any special reason that you used the pSB1C3_SEG for inserting the PCR products? And i just saw that u digested the PCR product with PstI, is that just a mistake happend accidentally by writing it down?? Thanks a lot!(Bea)

Comment: VP2/3 will be separated from cap for n-terminal fusion

Investigator: Jessica
DNA sample was diluted 1:1000

P357 = pAAV_RC
P396 = pAAV_RC_CapIns_prepSDM clone 3

PCR progam:

Ingredients	Volume / µl	Volume / µl
5X Phusion HF buffer	10	10
10 mM dNTP mix	1	1
forward primer: O90	2,5	2,5
reverse primer: O120	2,5	2,5
DNA Template	0,78	2
DMSO	1	1
Phusion Polymerase	0,5	0,5
H₂O	31,72	30,5
Total volume	50	50

PCR program:

Cycles	Temperature / °C	Time / s
	98	60
8x	98	15
	61	25
	72	30
17x	98	15
	72	25
	72	30
1x	72	300
Hold	4

Digestion of plasmid backbone:
Plasmid used: pSB1C3_RFC_SEG (P107)
c (pSB1C3_RFC_SEG) = 185,6 ng/ µl

Components	vector Volume/µL
DNA	5,4
BSA (10x)	2
Buffer no. 4 (10x)	2
NgoMIV	1
SpeI	1
H₂O	9,6
Total volume	20

incubation @ 37 C for approx. 2h

1% agarose gel

Digestion of PCR product:

Components	PCR product Volume/µL
DNA	30
BSA (10x)	4
Buffer no. 4 (10x)	4
NgoMIV	1
SpeI	1
H₂O	-
Total volume	40

incubation @ 37 C for approx. 2,5h

Ligation:

1 µl T4 DNA ligase, 1 µl 10x Buffer, µl vector (P107), µl Insert (P357)/ µl Insert (P396).
Incubation time: over night

Sequencing results of pCerulean_VP1up_NLS_Affibody/mVenus/Histag

Investigator: Anna

Comment: All results were classified in agreement with Sven. They can be used for generating the full construct (with VPex3).

pCerulean_VP1up_NLS_Affibody: Insert ok, but there is a G -> C transition.

pCerulean_VP1up_NLS_mVenus: The NgoMIV restriction site is present, but as above, there is a G -> C transition. Third mutation downstream is a sequencing fault, there are clearly two peaks for C.

pCerulean_VP1up_NLS_Histag: Insert was succesfully cloned into p_Cerulean.

Biobrick production of leftITR-phTERT-beta-globin and leftITR_pCMV-beta-globin

Motivation: Production of fusion proteins

Investigator: Kira

Digestions of backbones and inserts: c (pSB1C3_leftITR_phTERT) = 179 ng/ µl
c(pSB1C3_leftITR_pCMV) = 231 ng/ul

Components	TERT	CMV	beta-globin
DNA	5,5	4,5	8,5
BSA (100x)	0,25	0,25	0,25
Buffer no. 4 (10x)	2,5	2,5	2,5
PstI-HF	0,5	0,5	0,5
SpeI	1	1	1
XbaI	0	0	1
H₂O	15,25	16,25	12,25
Total volume	25	25	25

incubation @ 37 C for approx. h

1% agarose gel

Ligation:

Quickligase was used

TERT-mix 9 ul (5,3 ul vector + 3,7 ul insert)
CMV-mix 9 ul (3,7 ul vector + 5,3 ul insert)

Quickligase 1ul
Quickligase Buffer 10 ul

Incubation @ RT for 10'

Transformation:
Transformation was performed according to the standard protocol w XL1B cells (thanks Achim!)

Transfection, Harvest viral particles and Transduction

Investigator: Kerstin

Transfection of 15x 6-well plates (each plate same treatment --> 10 µg each plasmid)

Harvest viral particles (from 06.09.2010) and Transduction of HT1080 cells

1.plate:

- 1.1 no virus
- 1.2 lITR_CMV_beta-globin_mVenus_rITR
- 1.3 lITR_CMV_beta-globin_mVenus_rITR
- 1.4 lITR_CMV_beta-globin_mVenus_rITR
- 1.5 lITR_CMV_beta-globin_mVenus_Hgh_rITR
- 1.6 lITR_CMV_beta-globin_mVenus_Hgh_rITR

2.plate:

- 1.1 no virus
- 1.2 lITR_CMV_mVenus_Hgh_rITR
- 1.3 lITR_CMV_mVenus_Hgh_rITR
- 1.4 lITR_CMV_mVenus_Hgh_rITR
- 1.5 lITR_CMV_beta-globin_mVenus_Hgh_rITR
- 1.6 lITR_CMV_beta-globin_mVenus_Hgh_rITR

Competent XL1-B cells

Investigator: Patrick

Just for you! ;-)Do you know the normal transformation efficiency of chemically competent cells? What is good, waht is bad?? Thanks!

Today i checked the mutual competent cells i made for their transformation ability. It is ok (>10^6 cells / µg DNA).
Now there are about 100 new aliquots (each 100 µl) ready to use.

Checking PCR amplified virus particles

Investigator: Bea

Comment: The virus particles purifed by using liquid chromatography ("ÄKTA")(containing which construct??) were PCR- amplified (dame primers were used as for the qPCR which were already tested and could be confirmed for proper working) in order to obtain insights into which collected fraction does contain our virus particels. Therefore the PCR amplified samples were loaded on an 2% agarose gel.

Virus particle containing following plasmid: pAAV_iGEM_mVenus
Liquid chromatography performed by: Volker
PCR amplification performed by: Stefan

Results:

Sorry mistake: The blue highlighted boxes represent the cell pellet, not the supernatant!

As it can be seen in the picture, the expected PCR-fragment size of 200 bp can be detected in each fraction. Fractions collected 6 to 11 of the supernatant after cell lysis and centrifugation and fractions collected 6 to 11 of the cell pellet after cell lysis and centrifugation. The fraction 20 of the cell pellet was aswell PCR amplified and loaded on the agarose gel. The postivie control (added the plasmids containing a CMV promoter which were sequenced and confirmed) is loaded (pCerulean P274) and the negative control which contained only harvested HT1080 cells (harvested, but whithout any transduction) can be seen in lane 16 and 17, respectively. Since the PCR has been performed with a low T_M and 35 cycles this could be the reason for the detection of the CMV promoter of the negative control HT1080. next time PCR should be carried out with only 25 cycles and a higher T_M= 60°C. But the detection of the CMV promoter by using a normal PCR does work.

Quickchange site directed mutagenesis of pAAV_RC_CapIns_prepSDM

Investigator: Stefan

Comment: In cellculture, pAAV with inserted synthesised Rep did not work out. It was suggested that this could be related to an insertion of a KpnI restriction site near a splicing site. To verify this, the restriction site needs to be removed and tested in cellculture again.

Ingredients:

Ingredients	Volume / µl
10x reaction buffer	2,5
dNTP	0,5
forward primer: O182	0,52
reverse primer: O183	0,52
DNA Template	2
QuikSolution Reagent	0,75
QuikChangeLightning Enzyme	0,5
H₂O	17,71
Total volume	25

PCR program:

Cycles	Temperature / °C	Time / s
	95	120
8x (step 2-4)	95	15
	60	10
	68	225
	68	600

After PCR, the product was incubated with DpnI at 37 °C for 10 minutes.

Transformation:

Transformation was performed according to standard protocol using XL10-Gold cells. Cells were plated on plate containing Ampicilin.

116. labday 10.09.2010

Trafo evaluation of CD-biobrick plate

Investigator: Kira

Compared to the last tries, the ligation with T4 ligase was much more successful. The plate containes colonies.

Picking clones of yesterdays ViralBrick ligations and of Jessicas ligation

Investigator: Achim

Seeding HT1080 cells

Investigator: Kerstin

Ten 6-well plates of HT1080 cells were seeded (Transduction at 11.09.2010)for testing the optimal concentration of ganciclovir

Test digestion of pSB1C3_SV40_Terminator_Clone1-3

Investigator: Anna

Samples	Insert	Clone	Concentration [ng/µl]
1	pSB1C3_SV40_Terminator	1	204,2
2	pSB1C3_SV40_Terminator	2	246,1
3	pSB1C3_SV40_Terminator	3	200,9

Test digestion:

components	Volume for each sample /µl
DNA	3
BSA (10x)	1
Buffer 4 (10x)	1
Enzyme HF	0,5
Enzyme HF	0,5
H2O	4
Total volume /µl	10

Incubation time: 1,5 h, Incubation temperature: 37°

Preparation of gel:

0,5 g Agarose, 50 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 40 minutes

Comment: As shown on the gel, the vector backbone was religated with CFP (size: ~730 bp). PCR and Cloning steps have to be repeated, because the wrong restrition enzymes were used.

Sequencing analysis of pSB1A2_eGFP_HQ and pAAV_RC_CapIns_noRep

Investigator: Stefan
pSB1A2_eGFP_HQ

Comment: To improve iGEMs eGFP, it will be converted into RFC25 Freiburg standard. Plasmid preped from retrafo was sent for sequencing to verify sequence.

pAAV_RC_CapIns_noRep

Comment: Since pAAV containing the synthetised Rep does not work out in cell culture an additional approach was performed inserting only the synthesized Cap and test this.

Mini-prep and test digestion of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks

Investigator: Kira

Mini-Prep was performed with 3 clones of each construct in order to continue with test digestion and sequencing.

Test digestion:

components	Volume for each sample /µl
DNA	2.5
BSA (10x)	2
Buffer 4 (10x)	2
Enzyme Xba	1
Enzyme PstI-HF	0,5
H2O	12
Total volume /µl	20

1% agarose gel

CMV_clone1 as well as hTERT_clone1 were sent for sequencing

117. labday 11.09.2010

Sequencing evaluation of leftITR_CMV_beta-gloin and leftITR_hTERT_beta-globin biobricks

Investigator: Kira

The sequencing reveals that both biobricks were seccessful cloned.

Comment: For the evaluation, beta-globin intron was extracted from the original beta-globin biobrick and checked against the sequencing data (no connection to the lab server at home. the 'single' biobricks are only data, which are saved on my mac)

FACS analysis, harvest viral particles and Transduction

Investigator: Kerstin

FACS analysis of Constructs with/without Hgh/ß-globin
Harvested viral particles of transfection from 08.09.2010
Transduction for testing the optimal concentration of ganciclovir

Mini-Preps of Z34C ViralBricks

Investigator: Anna

Recent update of ViralBricks: The hybridisation and fill-in reactions of the Z34C constructs were repeated with heat inactivation of the Klenow enzyme (see labday 04.09.10), without usable results. In addition, a second ligation of 453_Bap was done with succes. This time two new approaches of the fill-ins were prepared (labday 09.09.10), one by using the Klenow Buffer and one with the Standard NEB Buffer 4.

Buffer 4:

Samples	Insert	Clone	Concentration [ng/µl]
11.4.1	453_ Z34C	1	271,67
11.4.2	453_ Z34C	2	218,01
12.4.1	587_Z34C	1	199,88
12.4.2	587_Z34C	2	357,13
-	587_KO_Z34C	-	-
-	587_KO_Z34C	-	-
14.4.1	587_KO_Z34C_Spacer	1	172,76
14.4.2	587_KO_Z34C_Spacer	2	337,54

Klenow Buffer:

Samples	Insert	Clone	Concentration [ng/µl]
11.1 K	453_ Z34C	1	220,89
11.2 K	453_ Z34C	2	176,34
12.1 K	587_Z34C	1	198,73
12.2 K	587_Z34C	2	182,64
13.1 K	587_KO_Z34C	1	186,50
13.2 K	587_KO_Z34C	2	158,51
14.1 K	587_KO_Z34C_Spacer	1	182,25
14.2 K	587_KO_Z34C_Spacer	2	180,06

Mini-Preps of separated VP2/3 for N-terminal fusion

Investigator: Anna

P357 = pAAV_RC
P396 = pAAV_RC_CapIns_prepSDM clone 3

Samples	Clone	Concentration [ng/µl]
357.1	1	290,74
357.2	2	282,97
357.3	3	307,63
396.1	1	302,04
396.2	2	314,58
396.3	3	298,77

Update and next steps of daily labwork

Investigators: Bea, Stefan

Preparation of pAAV_RC construct

We inserted the synthesized “rep” gene into the pAAV_RC construct with the four mutated restriction sites PstI (positions 310 and 4073), SalI (position 1239) and BamHI (position 856). The construct was tested in cell culture and no YFP expression of the vectorplasmid could be detected. Therefore, several new approaches were conducted.

The first approach was to remutate the only “unwanted” restriction site(KpnI) in the supposed regulatory region of the rep gene. The basic idea was that we delete the KpnI site because KpnI was in the 453 loop in which we could insert additional small motifs. But, this idea was dismissed after some more research. Therefore the KpnI restriction site does not have to be deleted in the rep sequence. This construct will be tested in cell culture.
Second, the experiment conducted in cell culture will be repeated with the same construct in order to exclude errors and mistakes related to the realization in cell culture.
Since we cannot be sure if the inserted rep containing the remutated KpnI restriction site still works in cell culture, we decided to insert the synthesized “cap” into the already tested and confirmed pAAV_RC containing only the four mutated restriction sites (see above). This construct will be tested in cell culture aswell.

Overview:

no plasmid number yet	P158	P250	P396	P413
pAAV_RC (4fold mutated, ins rep, SDM KpnI, inserted “cap”)	pAAV_RC (4-fold mutated) P158	pAAV_RC (4fold. Inserted “rep”)P250	pAAV_RC (4fold mutated, rep, cap)P396	pAAV_RC ((4fold mutated, cap) P413
not yet tested in cell culture	tested in cell culture and works	tested in cell culture, does not work. Experiment will be repeated	will not be tested in cell culture -> was used for SDM in rep gene.	has to be tested P413
desired construct. Clones were picked today!!	contains many restriction enzymes. Is not the construct of choice	If second experiment fails again, will not be used then.	was only used for SDM in rep gene.	If cell culture experiments work --> synthesized cap still functions properly.

N-terminal fusion

The constructs for the VP1 insertion and the Vp-2 fusion are ready for the next cloning step which means that we only need to fuse the VP2/3 PCR construct to the engineered plasmids containing different approaches. Two strategies will be followed until now:

The first strategy is to PCR amplify the VP2/3 construct from the pAAV_RC (4fold mutated, P357 in order to fuse the working VP2/3 contruct to the targeting approaches.
was performed by Jessica (9.9.10), waiting for results
The second strategy is to use the pAAV_RC (4fold mutated, ins rep, cap, P396) to PCR amplify the desired construct VP2/3.
was performed by Jessica (9.9.10), waiting for results
The next step is to PCR amplify the VP2/3 aswell from the construct pAAV_RC (4fold mutated, ins rep, SDM KpnI, inserted “cap”)
plasmid should be ready tomorrow (12.9.10), can be performed afterwards

Comment (Hanna): We want to amplify VP2/3 from pAAV_RC (it's indifferent, whether the PCR is done with the "WT"-pAAV_RC or with the pAAV_RC- 4fold mutated... (the mutations are in rep!) Further on, we want to PCR amplify VP2/3 from pAAV_RC_capins and from pAAV_RC_capIns_HSPG-KO!!! Did you mean that?

Yes you are right! Thanks! We should keep that in mind, and do not forget about the HSPG knockout!!! (Bea) ;-)

Additionally, a site-directed mutagenesis has to be performed in order to obtain the plasmids which can be co-transfected with the modified construct containing the targeting molecules. Aim is, to knockout VP1 for VP1-insertion approaches and VP2 (knockout) for VP-2 fusion. This site-directed mutagenesis has to be performed at several constructs:

P357 --> pAAV_RC (4fold mutated)
will be performed tomorrow by Bea/Stefan (12.9.10)
P396 --> pAAV_RC (4fold mutated, ins rep, cap)
will be performed tomorrow by Bea/Stefan (12.9.10)
P413 --> pAAV_RC (4fold mutated, ins cap)
will be performed tomorrow by Bea/Stefan (12.9.10)
pAAV_RC (4fold mutated, ins rep, SDM KpnI, inserted “cap”) --> since no Mini-preps have been prepared until now, no plasmid number is available.
dependant on test digestion performed tomorrow (12.9.10), if it works out, it will be performed tomorrow (12.9.10) as well

118. labday 12.09.2010

Mini-Prep and test digestion of pAAV_RC_RepCapIns_SDMKpnI

Investigator: Stefan

Glycerol stocks were prepared:

B371 = pAAV_RC_RepCapIns_SDMKpnI clone 1
B372 = pAAV_RC_RepCapIns_SDMKpnI clone 2
B373 = pAAV_RC_RepCapIns_SDMKpnI clone 3

Mini-Prep was performed according to the standard protocol

P431 = pAAV_RC_RepCapIns_SDMKpnI clone 1 = 362,21 ng/µl
P432 = pAAV_RC_RepCapIns_SDMKpnI clone 2 = 344,49 ng/µl
P433 = pAAV_RC_RepCapIns_SDMKpnI clone 3 = 331,30 ng/µl

Constructs were digested with KpnI for 70 minutes at 37°C, as controls the following constructs were used:

pAAV_RC_1.2SDMSalI (P158)
pAAV_RC_CapIns_prepSDM (P396)
pAAV_RC_CapIns_noRep (P413)

components	pAAV_RC_RepCapIns_SDMKpnI clone 1-3	pAAV_RC_1.2SDMSalI (P158)	pAAV_RC_CapIns_prepSDM (P396)	pAAV_RC_CapIns_noRep (P413)
DNA	1	1	1	1
Buffer 1 (10x)	1	1	1	1
BSA (10x)	1	1	1	1
KpnI	0,5	0,5	0,5	0,5
H₂O	6,5	6,5	6,5	6,5
Total volume	10	10	10	10

Preparation of gel:

0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes

Freiburg10 test digestion pAAV RC RepCapIns SDMKpnI.jpg

Comment: Test digestions look fine. Work will be continued with clone 2 (P432) which will be sent for sequencing tomorrow as well.

Test digestion of pSB1C3_VP2/3 needed for N-terminal fusion

Investigator: Bea

Comment: Test digestion of the two different approaches Jessica started on Friday has been performed. The general idea of the approach is to obatain VP2/3 cap genes (which means that the 137 amino acids of VP1 are missing) which then can be fused to the targeting molecules for the VP1 insertion and VP3 fusion approaches.
Note: P257_1 - 3 means that the pCR has been carried out with this construct. Same can be pbserved with the plasmid number P296.

Test digestion has been carried out using following enzymes:

Enzyme 1: PstI-HF
Enzyme 2: NgomIV

	Mastermix/µL	P357_1/µL	P357_2/µL	P357_2/µL	P396_1/µL	P396_2/µL	P396_2/µL
DNA	-	2	2	2	2	2	2
BSA (10x)	-	2,5	2,5	2,5	2,5	2,5	2,5
Buffer 4 (10x)	10,5
Enzyme NgoMIV	3,5
Enzyme PstI	3,5
H2O	-	10,5	10,5	10,5	10,5	10,5	10,5
Total volume	15	15	15	15	15	15	15

Test digestion was carried out for 55 minutes at 37°C.
The samples were loaded on a 1% agarose gel

Loading plan:

M   P357_1   P357_2   P357_3   P396_1   P396_2   P396_3

Results:

"P357" = pSB1C3_RC: 304bp , 1651bp , 2061bp
"P396"= pSB1C3_RC_ins_rep and cap: 1951bp , 2061bp

Freiburg10 Test digest pSB1C3 VP2-3 12.09.10.png

Quickchange site directed mutagenesis of pAAV_RC: VP1/VP2 knockout

Investigator: Stefan

Comment: A site-directed mutagenesis has to be performed in order to obtain the plasmids which can be co-transfected with the modified construct containing the targeting molecules. Aim is to knockout VP1 for VP1-insertion approaches and VP2 (knockout) for VP-2 fusion. This site-directed mutagenesis has to be performed at several constructs:

Knock-out of VP1:

pAAV_RC_1.2SDMSalI (P158)
pAAV_RC_CapIns_prepSDM (P396)
pAAV_RC_CapIns_noRep (P413)
pAAV_RC_RepCapIns_SDMKpnI (P432)

Knock-out of VP2:

pAAV_RC_1.2SDMSalI (P158)
pAAV_RC_CapIns_prepSDM (P396)
pAAV_RC_CapIns_noRep (P413)
pAAV_RC_RepCapIns_SDMKpnI (P432)

Used primers:
Knock-out of VP1:

primer forward: VP1-M1L_for (O170)
primer reverse: VP1-M1L_rev (O171)

Knock-out of VP2:

primer forward: VP2-ko_for (O165)
primer reverse: VP2-ko_rev (O166)

Used plasmid and water per approach:

plasmid no	used plasmid (same for both approaches) (1:100) / µl	VP1-ko: used water / µl	VP2-ko: used water / µl
P158	0,5	18,99	19,23
P396	2	17,49	17,73
P413	3	16,49	16,73
P432	3	16,49	16,73

Ingredients:

Ingredients	VP1-ko approach Volume / µl	VP2-ko approach Volume / µl
10x reaction buffer	2,5	2,5
dNTP	0,5	0,5
forward primer:	O170 - 0,38	O165 - 0,50
reverse primer:	O171 - 0,38	O166 - 0,52
DNA Template	see above	see above
QuikSolution Reagent	0,75	0,75
QuikChangeLightning Enzyme	0,5	0,5
H₂O	see above	see above
Total volume	25	25

PCR program:

Cycles	Temperature / °C	Time / s
1	95	120
2 8x(step 2-4)	95	15
3	60	10
4	68	225
5	68	600

After PCR, the products were incubated with DpnI at 37 °C for 10 minutes.

Transformation:

Transformation was performed according to standard protocol using XL10-Gold cells. Cells were plated on plate containing Ampicilin.

Comment: There were not enough cells for all approaches. Therefore, only transformation of approach 5 and 8 were performed using 45 µl of cells. The other approaches were conducted with 30 µl.

119. labday 13.09.2010

Biobrick assembly: Cloning VP2/3 and VP2/3_ins_Cap to several targeting approaches

Investigator: Stefan

Comment: Goal of the experiment is to fuse VP2/3 or VP2/3_ins_Cap to pCerulean_VP1up_NLS_Affibody, pCerulean_VP1up_NLS_ Histag and pCerulean_VP1up_NLS_mVenus.

Digestion of the constructs:
Inserts:

pSB1C3_VP2/3 (P440) c= 290,74 ng/µl
pSB1C3_VP2/3_ins_cap (P444) c= 314,58 ng/µl

Vectors:

pCerulean_VP1up_NLS_Affibody (P422) c= 390,68 ng/µl
pCerulean_VP1up_NLS_Histag (P424) c= 439,57 ng/µl
pCerulean_VP1up_NLS_mVenus (P426) c= 416,59 ng/µl

Restriction enzymes:
The inserts were digested using SpeI and NgoMIV, the vectors were digested using AgeI and SpeI.

Components	P440 /µL	P444 /µL	P422 /µL	P424 /µL	P426 /µL
DNA	5	4,5	6,5	5,5	6
BSA (10x)	2	2	2	2	2
Buffer 4 (10x)	2	2	2	2	2
Enzyme 1	1	1	1	1	1
Enzyme 2	1	1	1	1	1
H₂O	9	9,5	7,5	8,5	8
Total volume	20	20	20	20	20

Gel:
0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 90 Volt, running time: ~90 minutes

Freiburg10 pCerulean VP1up NLS targeting VP2 3.jpg

Gel-Extraction:
Marked bands on the gel were cut out. Gel extraction was performed according to protocol.

T4 Ligation:

All ligation approaches were performed using 1 µl of vector and 7 µl of insert. 1 µl of T4 ligation buffer and 1 µl of T4 ligase were used.

Transformation:

Transformation was performed according to standard protocol using BL21 cells and plates containing Kanamycin.

Biobrick production: P40 & SV40

Investigator: Patrick

So did u repeat the experiment?? Or are u ging to repeat it tomorrow?? (You know who I am ;-)

P158 (398 ng/µl) was used as P40 template and P230 (367 ng/µl) as SV40 template. Both of them were diluted 1:400.

SV40:

Ingredients	volume
5X Phusion HF buffer	10 µl
10 mM dNTP mix	1µl
forward primer: O158	2,5 µl
reverse primer: O159	2,5 µl
DNA Template	1 µl
DMSO	0 µl
Phusion Polymerase	0,5 µl
H₂O	32,5 µl
Total volume	50 µl

Cycles	Temperature	Time
	98°C	1
8x	98°C	15"
	59°C	25"
	72°C	7"
17x	98°C	15"
	67°C	25"
	72°C	7"
1x	72°C	5'
Hold 4°C

P40:

Ingredients	Volume / µl
5X Phusion HF buffer	10
10 mM dNTP mix	1
forward primer: O152	2,5
reverse primer: O153	2,5
DNA Template	1
DMSO	-
Phusion Polymerase	0,5
H₂O	32,5
Total volume	50

PCR program:

Cycles	Temperature / °C	Time / s
	98	60
8x	98	15
	61	25
	72	10
17x	98	15
	69	25
	72	10
1x	72	300
Hold	4

Expected size of the PCR products: P158/p40: 180 bp, P230/SV40: 230 bp.

Freiburg10 patrick p40 sv40 modified2.jpg

The visible bands do not have the expected size. These are probably amplified primers. The experiment has to be repeated.

Tobi proposed the following changes:

Use 1,5 ng template for p40 and SV40.

p40 PCR program:

Cycles	Temperature / °C	Time / s
	98	30
10x	98	10
	58	25
	72	10
15x	98	10
	66	25
	72	10
1x	72	300
Hold	4

SV40 PCR program:

Cycles	Temperature	Time
	98°C	30"
10x	98°C	10"
	56°C	25"
	72°C	10"
15x	98°C	10"
	64°C	25"
	72°C	10"
1x	72°C	5'
Hold 4°C

Test digestion of ViralBrick ligation and pTert BioBrick assembly

Investigator: Achim, Anna

Our Samples were digested with EcoRI and PstI again.
We used negative controls pSB1C3_Bla, containing a ~800bp insert and pSB1C3_SEG_linker, containing a ~160bp insert.

Additionally, a new test digestion of the pTert Biobrick assembly was prepared,this time with DrdI, an enzyme that should cut once in the insert and once in the vector. Expected sizes: ~2000 & 2500bp

Test Digestion ViralBricks:

Comparison with control fragments shows that most clones dont contain a correct insert. (Insert sizes should be about 230 bp.) We sent sample 11.2 (Buffer 4) and 11.1 (Klenow buffer) for sequencing

Test Digestion Assembly

2 distinct bands are visible, the expected sizes of 2000 and 2500 bp match with the ladder.

PCR of RepCap/Cap

Investigator: Jessica

Dear Jessy, would you please always comment and explain what you're doing - so that other persons also have a chance to understand what you've done!

DNA sample was diluted 1:100

P158 = pAAV_RC_1.2 SDM SalI
P396 = pAAV_RC_CapIns_prepSDM clone 3
P413 = pAAV_RC_CapIns_noRep clone 1
P432 = pAAV_RC_RepCapIns_SDMKpnI clone 2

PCR progam:
for Rep Cap

Ingredients	Volume P158 / µl	Volume P396/ µl	Volume P413/ µl	Volume P432/ µl
5X Phusion HF buffer	10	10	10	10
10 mM dNTP mix	1	1	1	1
forward primer: O93	2,5	2,5	2,5	2,5
reverse primer: O120	2,5	2,5	2,5	2,5
DNA Template	0,25	0,20	0,32	0,30
DMSO	-	-	-	-
Phusion Polymerase	0,5	0,5	0,5	0,5
H₂O	33,25	33,30	33,18	33,20
Total volume	50	50	50	50

no DNA on the gel...

for Cap

Ingredients	Volume P158 / µl	Volume P413/ µl
5X Phusion HF buffer	10	10
10 mM dNTP mix	1	1
forward primer: O89	2,5	2,5
reverse primer: O120	2,5	2,5
DNA Template	0,25	0,32
DMSO	-	-
Phusion Polymerase	0,5	0,5
H₂O	33,25	33,18
Total volume	50	50

Why did you use two times the same PCR program and the same primer for different approaches?

PCR program:

Cycles	Temperature / °C	Time / s
	98	60
8x	98	15
	60	25
	72	70
17x	98	15
	69	25
	72	70
1x	72	300
Hold	4

Digestion of plasmid backbone:
Plasmid used: pSB1C3_VCK_Bla (P320)
c (pSB1C3_VCK_Bla) = 408,0 ng/ µl

Components	vector Volume/µL
DNA	2,45
BSA (10x)	2
Buffer no. 4 (10x)	2
NgoMIV	1
SpeI	1
H₂O	11,55
Total volume	20

incubation @ 37 C for approx. 2h

1% agarose gel

Digestion of PCR product:

Components	PCR product Volume/µL
DNA	30
BSA (10x)	4
Buffer no. 4 (10x)	4
NgoMIV	1
SpeI	1
H₂O	-
Total volume	40

incubation @ 37 C for approx. h

Ligation:

1 µl T4 DNA ligase, 1 µl 10x Buffer, µl vector (P320), µl Insert (P158)/ µl Insert (P413).
Incubation time:

Miniprep and test digestion of CD biobricks

Investigator: Kira

Components	sample Volume/µL
DNA	4
BSA (10x)	2
Buffer no. 4 (10x)	2
XbaI	1
AgeI-HF	0,5
H₂O	10,5
Total volume	20

incubation @ 37 C for approx. 2h

1% agarose gel

BioBrick ^TM assembly of pSB1C3_leftITR_promoter_betaglobin to several GOIs

Investigator: Bea

Comment: Since we only assembled the vecotrplasmid with the CMV promoter and mVenus_YFP as gene of interest other approaches have to be conducted, especially the vectorplasmid which contains the tumorspecific promoter phTERT with the gene of interest mGMK_TK30. These approaches were started by Kira some days ago, and will now be continued. This time we are fusing the extisting and confirmed constructs pSB1C3_leftITR_CMV_betaglobin and pSB1C3_leftITR_phTERT_betaglobin to the gene of interests mVenus_YFP and mGMK.

Digestion of the constructs:

P434 = pSB1C3_leftITR_CMV_betaglobin c=331ng/µL
P434 = pSB1C3_leftITR_phTERT_betaglobin c=331ng/µL

Components	v_P434 /µL	v_P437 /µL
DNA	4,5	4,5
BSA (10x)	2	2
Buffer no. 4 (10x)	2	2
SpeI	1	1
PstI-HF	1	1
H₂O	9,5	9,5
Total volume	20	20

Digestion of the three targeting approaches:

P81 = pAAV_iGEM_mGMK_TK30 --> approach will not be proceeded becuase the iGEM restriction sites cut in the vctor as well and therefore the desired fragments cannot be isolated with used iGEM restriction sites.!!!
P195 = pSB1C3_mGMK c=258ng/µL
P229 = pAAV_iGEM_mVenus c=486 ng/µL

Components	v_P81 /µL	v_P195 /µL	v_P229 /µL
DNA	7,8	12,3	4,1
BSA (10x)	2,5	2,5	2,5
Buffer no. 4 (10x)	2,5	2,5	2,5
XbaI	1	1	1
PstI-HF	1	1	1
H₂O	10,2	12,3	13,9
Total volume	25	25	25

Loading plan:

M    P434  P437  P81  P195  P229

Results of preparative agarose gel:
Expected sizes:

Freiburg10 Digestion of pSB1C3 CMV promoter bglob GOI.jpg

After the gel extraction (the fragments cut out of the gel were labeled) has been performed the ligation was carried out.

Ligation:

v_=P434=5,61µL
v_=P195=2,39µL

v_=P437=4,77µL
v_=P195=3,32µL

v_=P437=4,17µL
v_=P229=3,83µL

After mixing insert and vector into a tube, add 1µL T4-ligase 10x buffer and 1µL ligase to the DNA mix and incubate for 40 minutes at room temperature.
Transform XL1-B cells, and plate them on agar containing chloramphenicol.

Next steps: Picking clones of the constructs, prepare Mini-prep and finally fuse them to the existing construct pSB1C3_hGH_rightITR!

Sent the construct pSB1C3_VP2/3 and pSB1C3_VP2/3 for sequencing

Investigator: Bea

Comment: The construct which has been produced (experiment started on friday) will be sequenced today. The construct were named: P357 1- 3 and P396 1-3. After preparing for sequencing the plasmids were renamed and received a plasmid number aswell.

P357_1 = P440
P357_2 = P441
P357_3 = P442
P396_1 = P443
P396_3 = P443
P396_3 = P445

The two constructs which have been sent for sequencing are:

P440 = BK_1, used primers: VR-2 and VF-2
P444 = BK_2, used primers: VR-2 and VF-2

Fusion of VP2/3 to C-terminus of Zegfr:1907_"Linker", CFP_MiddleLinker and 6xHis_MiddleLinker construct

Investigator: Hanna

UPDATE: The Zegfr:1907 Affibody was successfully fused to the different linker (Short Linker, Long Linker, Middle Linker and SEG Linker) in order to find out with which linker targeting functions best. In addition to that, CFP (Imaging) and the His-Tag (=6xHis, Purification) was fused to the Middle Linker. These constructs were cloned into the pCerulean plasmid (in order to be driven by the CMV promoter + SV40 terminator).
The last steps in the VP2-N-terminal fusion approach include the knockout of the VP2 start codon in pAAV_RC (different constructs: 4-fold mutated, 4-fold mutated with Cap-insert, 4-fold mutated with Rep/Cap-insert/KpnI-backmutation) and fusion of the VP2/3 ORF to the C-terminus of the linker.

SDM of the VP2 start codon (of the unmodified and Cap-insert sequence) was already performed by Stefan. After the Cap-insert variant has been tested in cell culture, the HSPG-KnockOut motif will be cloned into Cap and also used for BioBrick production via PCR. This fragment will also be used for fusion to the C-terminus of the Linker-Library+Affibody and the purification (6xHis) and Imaging (CFP, better: GFP) approaches. Therefore pAAV_RC_Rep/Capins_KpnI-backmutation_HSPG-KO (if Rep-KpnI-backmutation works!) will be also used for SDM of VP2 start codon.

Today VP2/3 (unmodified) and VP2/3-Cap-ins will be fused to the C-terminus of the linker. These approaches will be co-transfected with the referring pAAV_RC_VP2-KO constructs and therefore tested in cell culture on wednesday.

Practical Cloning:

- Vector: pCerulean_CFP_MiddleLinker (P407), pCerulean_Zegfr:1907_MiddleLinker (P408), pCerulean_Zegfr:1907_SEG (P409), pCerulean_Zegfr:1907_ShortLinker (P410), pCerulean_Zegfr:1907_LongLinker (P412), pCerulean_6xHis_MiddleLinker (P374)
- Insert: pSB1C3_VP2/3 (P440), pSB1C3_VP2/3_ins_cap (P444)
- new vector name: pCerulean_CFP_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_SEG_VP2/3, pCerulean_Zegfr:1907_ShortLinker_VP2/3, pCerulean_Zegfr:1907_LongLinker_VP2/3, pCerulean_6xHis_MiddleLinker_VP2/3 AND pCerulean_CFP_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_SEG_VP2/3-insCap, pCerulean_Zegfr:1907_ShortLinker_VP2/3-insCap, pCerulean_Zegfr:1907_LongLinker_VP2/3-insCap, pCerulean_6xHis_MiddleLinker_VP2/3-insCap
buffer used:4

Comment Because the VP2/3 frament has approximately the same size as the backbone pSB1C3 it may be difficult to separate them on the agarose gel. Therefore a single cutter enzyme (SacI) was chosen to digests the backbone. This enzyme also works in buffer 4 + BSA and needs to be incubated at 37°C.

- Vector: AgeI-HF, SpeI
- Insert: NgoMIV, SpeI, SacI

Digestion

components	volume of vector /µl	volume of insert /µl
DNA	3.75	8.3
BSA (10x)	2	3
Buffer 4(10x)	2	3
AgeI-HF	1	-
NgoMIV	-	1
SpeI	1	1
SacI	-	1
H₂O	10.25	12.7
Total volume	20	30

Incubation: 2 h

Comment_ Because we are all hardworking cloners :) there was not enough AgeI left for all 6 reactions (just 3: P374, P407, P408). Unfortunately the lab stock AgeI was also empty. Therefor we had to "lend" it from the Baumeister group - which is already from "Fermentas" and therefore also needs Fermentas-buffer. Nevertheless we checked the pH from buffer 4 with the one needed for the Fermentas-AgeI and decided to try using this AgeI in buffer 4.

Agarose-Gel:

0.8 g and 0.4 g Agarose, 50 mL TAE (0.8 % and 1.5 %), 3 µL GELRED, at 90 Volt, running time:60 minutes Comment: Unfortunately the gels were interchanged so that the vector was loaded onto the 1.5% gel.

Sample	Sample/µl]	Loading dye (6x)/µl	Expected size 1	Expected size 2	Expected size 3
P407	20 µl	4 µl	~ 4170 bp	-	-
P408	20 µl	4 µl	~ 4170 bp	-	-
P409	20 µl	4 µl	~ 4170 bp	-	-
P410	20 µl	4 µl	~ 4170 bp	-	-
P412	20 µl	4 µl	~ 4170 bp	-	-
P374	20 µl	4 µl	~ 4170 bp	-	-
P440	30 µl	6 µl	1900 bp	910 bp	1150 bp
P444	30 µl	6 µl	1900 bp	910 bp	1150 bp

Marker: GeneRuler ladder mix

	Marker /µL	Sample Jessy /µl	Sample P407 /µl	Sample P408 /µl	Sample P409 /µl	Sample P410 /µl	Sample P412 /µl
Lane	5.5		24	24	24	24	24

	Sample P374 /µL	Sample P440 /µl	Sample P444 /µl	Marker /µl
Lane	24	36	36	5.5

Gel extraction

Gel measurement:

Sample	Weight
P407	150 mg
P408	180 mg
P409	230 mg
P410	220 mg
P412	170 mg
P374	180 mg
P440	210 mg
P444	180 mg

Ligation

Construct	Vector (µl)	Insert (µl)
407 + 440	4.6	3.4
407 + 440	4.98	3.02
408 + 440	4.14	3.86
408 + 444	4.53	3.47
409 + 440	4.18	3.82
409 + 444	4.57	3.43
410 + 440	3.6	4.4
410 + 444	3.99	4.01
412 + 440	3.6	4.4
412 + 444	3.99	4.01
374 + 440	3.23	4.77
374 + 444	3.61	4.39

120. labday 14.09.2010

Test Digestion of remaining ViralBrick clones

Investigator: Achim

None of the samples contained inserts of the expected size. (Either religated vector or BLA). Samples were discarded. New ligation tomorrow.

ViralBrick sequencing results

Investigator: Achim

11.2, Buffer 4

11.1, Klenowbuffer

The two plasmids contain Z34C, but are incomplete in the 453 upstream region. There could be several reasons for this: Incompletely synthesized oligos, incomplete fill-in reactions...

Tomorrow we're going to try another repetition of the hybridisation and ligation, this time with dephosohorized vector - that should prevent the growth of clones containing religated vector. We will also use new Klenow fragment and try two parallel ligations: Quickligation & T4 ligation over night.

Retrafo with sr39_rightITR (Mr.Gene)

Investigator: Bea

Comments: We received the sr39 (mutant verstion of the wt thymidinkinase) and wt rightITR ordered from Mr. Gene today. In order to use and clone the insert into the desired sequences a retrafo has to be performed.

Re-transformation:

Construct used:
We reveived 5 µg (lyophilzied). These were resuspended in 100 µL H₂O and vortexted.
c = 50 ng/µL
c = Plasmid need to be diluted in order to obatain the final 500pg
Dilution: 1:100
100 µL aliquot of XL1-B were thawed on ice. The proper amount of 1µL was added to the cells.
Transformed cells were plated on LB plates containing kanamycin

Picking clones of pSB1C3_leftITR_promoter_betaglobin_GOI

Investigator: Bea

Comments: Clones were picked from the approach condutced yesterday.

pSB1C3_leftITR_CMV_betaglobin_mGMK
pSB1C3_leftITR_phTERT_betaglobin_mGMK
pSB1C3_leftITR_phTERT_betaglobin_mVenus

Additionally, pHelper (Glycerol stock) was inoculated in 30 mL containing ampicillin.

3^rdrepetition of SV40 terminator (hopefully the last one ;-))

Investigator: Bea

Comments: The last repetition of the PCR for obtaining the SV40 fragment did not work out because wrong primers were used therefore the PCR will be carried out once again.

Preparations:
A 1:1000 dilution of the plasmid P230 (pEGFP_C1) which contains the desired SV40 polyadenlyation site will be prepared. This fragment will be extracted and cloned into pSB1C3 iGEM standard plasmid.

PCR protocol:

Ingredients	volume
5X Phusion HF buffer	10 µl
10 mM dNTP mix	1µl
forward primer: O158	2,5 µl
reverse primer: O159	2,5 µl
DNA Template	3,7 µl
DMSO	0 µl
Phusion Polymerase	0,5 µl
H₂O	29,8 µl
Total volume	50 µl

The following PCR programm was used. Idea is that in the first 8 cycles the primer can only bind to the template added in to the reaction mix. The following 17 cycles afterwards allow the annealing of the complete primer containing prefix and suffix with its restriciton sites.

Cycles	Temperature	Time
	98°C	1
8x	98°C	15"
	59°C	25"
	72°C	7"
17x	98°C	15"
	67°C	25"
	72°C	7"
1x	72°C	5'
Hold 4°C

The PCR product (after PCR purification) and the plasmid vector pSB1C3_CFP (P51.2) will be digested with the same restriction enzymes and afterwards loaded on a 1% agarose gel:

XbaI
PstI

Components	v_{PCR product SV40} /µL	v_P51.2 /µL
DNA	20	9
BSA (100x)	0,3	0,2
Buffer no. 4 (10x)	3	2
SpeI	1	1
PstI-HF	1	1
H₂O	4,7	6,8
Total volume	30	20

After digestion of the two constructs they were loaded on a 1% agarose gel.

M    PCR product SV40    pSB1C3_CFP

Expected sizes of the two digested constructs are:

PCR product SV40 terminator: 255bp
pSB1C3: 2053bp
pSB1C3: 756bp

Results:

PCR fragment was cut out after 20 minutes running the gel. The vector was cut out after 40 minutes.

After gel extraction has been performed, the ligation was carried out.

Ligation:

v_=pSB1C3=7,7µL
v_=SV40=0,3µL

The ligation mix was transformed into XL1-Blue cells and plated on agar plates containing chloramphenicol.

Next steps:
Picking clones and perform Mini-Prep. This construct can then be submitted to the parts registry after sequencing.

Sequencing analysis from pSB1C3_VP2/3 and pSB1C3_VP2/3_ins_cap

Investigator: Bea

Comment: The constructs which were sent for sequencing are the plasmids which were PCR amplified from the pAAV_RC and cloned into the pSb1C3. Purpose of the PCR amplification was to clone the fragement to the N-terminal targeting approaches.

P440 = pSB1C3_VP2/3 = BK_1
Used primers:

VR-2
VF-2

Results:

Freiburg10 Seq analysis P440 BK1 VF2 13.09..jpg

P444 = pSB1C3_VP2/3_ins_cap = BK_2
Used primers:

VR-2
VF-2

Results:

Freiburg10 Seq analysis P444 BK2 VF2 13.09..jpg

´

Midiprep of pAAV_RC_CapIns_NoRep; pAAV_RC_RepCapIns_SDMKpnI

Investigator: Chris W.
The Midiprep was performed according to the standard protocol yieldung the following concentrations:

pAAV_RC_CapIns_NoRep: 387,05 ng/µl, labeled as P467
pAAV_RC_RepCapIns_SDMKpnI: 1157.58 ng/µl labeled as P468

Sequencing analysis from pAAV_RC_RepCapIns_SDMKpnI

Investigator: Stefan

Comment: Last week's SDM to remutate the KpnI restriction site in Rep needed to be sequenced.

P432 = pAAV_RC_RepCapIns_SDMKpnI = AM_3
Used primers: 1250 for

Results:

Freiburg10 Sequencing pAAV RC RepCapIns SDMKpnI.jpg

Repetition of the fusion of VP2/3 to C-terminus of Zegfr:1907_"Linker", CFP_MiddleLinker and 6xHis_MiddleLinker construct

Investigator: Anissa

COMMENT: The same approach which has been done yesterday, was repeated today, because gel-pictures showed no satisfying results.

Practical Cloning:

- Vector: pCerulean_CFP_MiddleLinker (P407), pCerulean_Zegfr:1907_MiddleLinker (P408), pCerulean_Zegfr:1907_SEG (P409), pCerulean_Zegfr:1907_ShortLinker (P410), pCerulean_Zegfr:1907_LongLinker (P412), pCerulean_6xHis_MiddleLinker (P374)
- Insert: pSB1C3_VP2/3 (P440), pSB1C3_VP2/3_ins_cap (P444)
- new vector name: pCerulean_CFP_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_SEG_VP2/3, pCerulean_Zegfr:1907_ShortLinker_VP2/3, pCerulean_Zegfr:1907_LongLinker_VP2/3, pCerulean_6xHis_MiddleLinker_VP2/3 AND pCerulean_CFP_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_SEG_VP2/3-insCap, pCerulean_Zegfr:1907_ShortLinker_VP2/3-insCap, pCerulean_Zegfr:1907_LongLinker_VP2/3-insCap, pCerulean_6xHis_MiddleLinker_VP2/3-insCap
buffer used:4

Comment Because the VP2/3 frament has approximately the same size as the backbone pSB1C3 it may be difficult to separate them on the agarose gel. Therefore a 0,8% gel was prepared and the samples run for 1,5 h. So the two bands (1900bp and 2200bp) will hopefully be seperated.

- Vector: AgeI-HF, SpeI
- Insert: NgoMIV, SpeI

Digestion

components	pSb1C3_Vp2/3 (2,5µg) P440 /µl /µl	pSb1C3_Vp2/3_capIns (2,5µg) P444 /µl
DNA	8,6	8
BSA (10x)	1,5	1,5
Buffer 4(10x)	1,5	1,5
speI	1	1
NgoMIV	1	1
SpeI	1	1
H₂O	1,4	2
Total volume	15	15

components	pCerulean_affib._shortlinker P410 (1µg)/µl	pCerulean_affib._middlelinker P408 (1µg)/µl
DNA	2	2,5
BSA (10x)	1,5	1,5
Buffer 4(10x)	1,5	1,5
speI	1	1
AgeI	0,75	0,75
SpeI	1	1
H₂O	8,25	7,75
Total volume	15	15

components	pCerulean_affib._longlinker P412 (1µg)/µl	pCerulean_affib._SEG P409 (1µg)/µl
DNA	2,4	2,4
BSA (10x)	1,5	1,5
Buffer 4(10x)	1,5	1,5
speI	1	1
AgeI	0,75	0,75
SpeI	1	1
H₂O	7,85	7,85
Total volume	15	15

components	pCerulean_CFP_middlelinker P407 (1µg)/µl	pCerulean_His-tag._middlelinker P374 (1µg)/µl
DNA	2,1	2,5
BSA (10x)	1,5	1,5
Buffer 4(10x)	1,5	1,5
speI	1	1
AgeI	0,75	0,75
SpeI	1	1
H₂O	8,15	7,84
Total volume	15	15

Incubation: 1,5 h

Comment: 0,75 µl AgeI were used because AgeI is not deluted.

Agarose-Gel:

0.4 g and 0,5g Agarose, 50 mL TAE (0.8 %, 1%), 3 µL GELRED, at 110 Volt, running time:1,5 h

Sample	Sample/µl]	Loading dye (6x)/µl	Expected size 1	Expected size 2	Expected size 3
P407	20 µl	4 µl	~ 4170 bp	-	-
P408	20 µl	4 µl	~ 4170 bp	-	-
P409	20 µl	4 µl	~ 4170 bp	-	-
P410	20 µl	4 µl	~ 4170 bp	-	-
P412	20 µl	4 µl	~ 4170 bp	-	-
P374	20 µl	4 µl	~ 4170 bp	-	-
P440	30 µl	6 µl	1900 bp	910 bp	1150 bp
P444	30 µl	6 µl	1900 bp	910 bp	1150 bp

Marker: GeneRuler ladder mix

	Marker /µL	Sample p410 /µl	Sample P408 /µl	Sample P412 /µl	Sample P409 /µl	Sample P407 /µl	Sample P374 /µl
Lane	3	19	19	19	19	19	19

	Marker /µl	Sample P444 /µl	Sample P440 /µl
Lane	3	19	19

Gel extraction

Gel measurement:

Sample	Weight
P407	180 mg
P408	160 mg
P409	90 mg
P410	190 mg
P412	230 mg
P374	190 mg
P440	120 mg
P444	130 mg

Ligation

Construct	Vector (µl)	Insert (µl)
407 + 440	1	3
407 + 440	1	3
408 + 440	1	3
408 + 444	1	3
409 + 440	1	3
409 + 444	1	3
410 + 440	1	3
410 + 444	1	3
412 + 440	1	3
412 + 444	1	3
374 + 440	1	3
374 + 444	1	3

Comment: Because the amount of the insert-DNA wouldn't sufficiend for all ligations, the amount of the totl DNA was reduced to 4 µl.

Transformation

Transformation was performed in Xl1 blue according the standard-protocol.

Mini-Prep and test digestion of multiple plasmids

Investigator: Stefan

Glycerol stocks were prepared:

B382 = pAAV_RC_1.2SDMSalI_VP1-ko clone 1
B383 = pAAV_RC_1.2SDMSalI_VP1-ko clone 2
B384 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 1
B385 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 2
B386 = pAAV_RC_CapIns_noRep_VP1-ko clone 1
B387 = pAAV_RC_CapIns_noRep_VP1-ko clone 2
B388 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 1
B389 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 2
B390 = pAAV_RC_1.2SDMSalI_VP2-ko clone 1
B391 = pAAV_RC_1.2SDMSalI_VP2-ko clone 2
B392 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 1
B393 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 2
B394 = pAAV_RC_CapIns_noRep_VP2-ko clone 1
B395 = pAAV_RC_CapIns_noRep_VP2-ko clone 2
B396 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 1
B397 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 2

B398 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 1
B399 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 2
B400 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 1
B401 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 2
B402 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 1
B403 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 2
B404 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 1
B405 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 2
B406 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 1
B407 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 2
B408 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 1
B409 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 2

Mini-Prep was performed according to the standard protocol

P449 = pAAV_RC_1.2SDMSalI_VP1-ko clone 1 c= 372,90 ng/µl
P450 = pAAV_RC_1.2SDMSalI_VP1-ko clone 2 c= 380,47 ng/µl
P451 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 1 c= 367,08 ng/µl
P452 = pAAV_RC_CapIns_prepSDM_VP1-ko clone 2 c= 387,34 ng/µl
P453 = pAAV_RC_CapIns_noRep_VP1-ko clone 1 c= 360,67 ng/µl
P454 = pAAV_RC_CapIns_noRep_VP1-ko clone 2 c= 353,62 ng/µl
P455 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 1 c= 353,85 ng/µl
P456 = pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 2 c= 349,39 ng/µl
P457 = pAAV_RC_1.2SDMSalI_VP2-ko clone 1 c= 308,46 ng/µl
P458 = pAAV_RC_1.2SDMSalI_VP2-ko clone 2 c= 342,85 ng/µl
P459 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 1 c= 386,43 ng/µl
P460 = pAAV_RC_CapIns_prepSDM_VP2-ko clone 2 c= 327,22 ng/µl
P461 = pAAV_RC_CapIns_noRep_VP2-ko clone 1 c= 373,94 ng/µl
P462 = pAAV_RC_CapIns_noRep_VP2-ko clone 2 c= 347,39 ng/µl
P463 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 1 c= 372,55 ng/µl
P464 = pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 2 c= 348,33 ng/µl

P465 = pSB1C3_SEG c= 208,32 ng/µl
P466 = pSB1C3_mGMK c= 224,62 ng/µl

P470 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 1 c= 53,04 ng/µl
P471 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3 clone 2 c= 114,24 ng/µl
P472 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 1 c= 170,16 ng/µl
P473 = pCerulean_VP1up_NLS_Zegfr:1907_VP2/3-Capins clone 2 c= 145,98 ng/µl
P474 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 1 c= 104,29 ng/µl
P475 = pCerulean_VP1up_NLS_6xHis_VP2/3 clone 2 c= 147,86 ng/µl
P476 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 1 c= 97,33 ng/µl
P477 = pCerulean_VP1up_NLS_6xHis_VP2/3-Capins clone 2 c= 97,94 ng/µl
P478 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 1 c= 133,61 ng/µl
P479 = pCerulean_VP1up_NLS_mVenus_VP2/3 clone 2 c= 107,60 ng/µl
P480 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 1 c= 45,80 ng/µl
P481 = pCerulean_VP1up_NLS_mVenus_VP2/3-Capins clone 2 c= 115,16 ng/µl

Test digestion:
No test digestion was performed for Plasmid numbers P449 to P466. P449 to P464 were obtained from a SDM to knock-out VP1 or VP2. There is no possibility to verify this from a test digestion. P465 and P466 were obtained from glycerol stock and therefore no test digestion is necessary. The other constructs were digested using EcoRI and SpeI for 40 minutes at 37°C.

components	P470-P481
DNA	2
Buffer 1 (10x)	1
BSA (10x)	1
EcoRI	0,5
SpeI	0,5
H₂O	5
Total volume	10

Preparation of gel:

0,4 g Agarose, 50 ml TAE (0,8%), 3 µl GELRED , at 110 Volt, running time: 45 minutes

Freiburg10 test digestion pCerulean VP1up NLS targeting VP2 3.jpg

Comment: it is still not clear whether or not the correct insert was cloned into pCerulean. Therefore, P475 and P473 will be sent for sequencing tomorrow using pEGFP_C2-RP as primer to verify VP2/3 insertion.

Sent for sequencing:
Primer used:
2800 reverse (O38)

P449
P451
P453
P455
P457
P459
P461
P463

121. labday 15.09.2010

New ViralBrick cloning attempt

Investigator: Achim

Yet another attempt at hybridizing and ligating the ViralBrick oligos into the pSB1C3_BLA. Changes:

Dephosphorylation of digested vector to prevent religation
New Klenow Fragment from Fermentas
Two parallel ligation approaches:
- Quickligation for 10 min
- T4 Ligation over night

Following steps were carried out:

Hybridisatzion of oligos
- 11:453 Z34C: 143 & 144
- 12:587 Z34C: 145 & 146
- 13:587 KO Z34C: 147 & 148
- 14:587 KO Z34C: 149 & 150

Condtitions as before:

2 µg DNA, (3,3 µl of each oligo)
2 µl Klenow buffer
0,1 µl dNTPs (1µl of 1:10 dilution of our aliquots)
9,8 µl H2O

Total volume: 20 µl

Hybridisation was carried out on the gradient cycler using the program "tkfill"

0,5 µl Klenow Fragment was added after the temperature reached 37°C. Klenow reaction was carried out for 1 h in the 37°C room.

Afterwards, the samples were purified with the Quiagen PCR Purification kit. Elution in 50 µl H2O. Nanodrop concentrations:

11: 47.08 ng/µl
12: 28.50 ng/µl
13: 37.69 ng/µl
14: 42.98 ng/µl

1 µl of SspI & SalI for sample 11/ BamHI & PvuII for samples 12,13,14 as well as 5,8 µl buffer 4 for each sample were added to the purified samples. Digestion for 3 h at 37 °C

The vector backbone pSB1C3_VCK_BLA (P320)was also digested with BamHI/PvuII and SspI/SalI for 3 h. Afterwards,the cut vector fragment was dephosphorylated with Antarctic Phosphatase for 1h at 37°C.

The oligos and the vector fragments were mixed with loading dye and run over a preparative gel (1%):

Oligos ran between 100 - 200 bp, the unhybridized oligo control ran a bit further.

Fragments were cut out, followed by gel extraction. Two ligations were prepared: T4-Ligation and Quickligation.

Quickligation: 10 min, followed by trafo. Plates were incubated overnight.

T4-Ligation: Overnight

Cloning VP2/3 approaches into pSB1C3_VCK vector

Investigator: Bea

Comment:Since the constructs VP2/3 and VP2/3_ins_cap were cloned in the plasmid pSB1C3 they need to recloned into the vector pSB1C3_VCK which have two deleted restrictions sites (PvuII ans SspI) . These restriction sites have to be single cutting enzymes in this case because we want to subclone the HSPG-knockout "motif" into the cap gene in order to obtain constructs for the N-terminal targeting approach. The recombinant virus particles basically then do not show the natural tropism for its primary receptor.

Digestion of the constructs:

P440 = pSB1C3_VP2/3 c=290 ng/µL
P440 = pSB1C3_VP2/3_ins_cap c=314ng/µL
P309 = pSB1C3_VCK_BLA c=408ng/µL

Components	v_P440 /µL	v_P444/µL	v_P309 /µL
DNA	5,5	5,5	2,5
BSA (10x)	4	4	3
Buffer no. 4 (10x)	4	4	3
EcoRI	1	1	1
AgeI	1	1	1
MISC	SacI: 1	MscI: 1	-
H₂O	23,5	23,5	19,5
Total volume	40	40	30

The expected fragments of the digested constructs were: The underlined fragments represent the fragments which correspond to the desired construct which can be used for ligation.

P440: 1950, 1158, 908bp
P444: 1950, 1347, 715bp
P309: 2067, 894bp

Loading plan:

M    P440    P444    P309

Results:

Freiburg10 pSB1C3 VCK VP2 3 15 09 2010.jpg

After gel extraction has been performed, the ligation was carried out.

Ligation:

v_=P444=5,5µL
v_=P309=2,5µL

The ligation mix was transformed into XL1-Blue cells and plated on agar plates containing chloramphenicol.

Next steps:
Picking clones and perform Mini-Prep. After the Mini-Preps have been performed these constructs can be used for inserting the HSPG knockout motif.

Colony PCR of CD colonies

Investigator: Kira

Comments: Since the last approach with the CD did not work, we performed a colony PCR in order to detect the positive clones which should contain the CD gene. We used the two SDM primers, originally used for deleting the iGEM restriction sites in the CD gene, which should only bind to the CD sequence.

15 colonies were picked from the plate and dispersed first into the PCR tube and then into DYT media as a backup in case of successful PCR reaction.

components	pro sample
10x Taq Buffer	1
MgCl (50mM)	0,1
dNTPs	0,2
Primer 1	0,2
Primer 2	0,2
Taq Polymerase	0,05
H₂O	8,25
Total volume	10

PCR program:

Cycles	Temperature	Time
	95°C	6 min
30x	95°C	25 sec
	63°C	30 sec
	68°C	1 min 6 sec
1x	68°C	5 min
Hold 4°C

pSB1C3_RFC25_CFP was used as a control sample

1% agarose gel

According to the gel, clone 3, 6, 7, 9, 11, 14 contain CD gene. These samples will be incubated over-night @ 37C.

Comment added on 17.09.2010 : We (Bea, Kira, Stefan and Tobi) wondered why sequencing of one of the positive clones of the colony PCR revealed that the construct did not contain the expected CD gene but CFP instead. Today, we realized that the colony PCR does not reveal only positive constructs which contain the cytosine deaminase. Problem is that we received the CD gene originating from the COD-BA gene of E. coli. Since we performed a colony PCR with XL1-B E. coli cells the used primers will bind to the genomic CD-gene aswell which could explain the positive clones in the gel, the negative results of seuqencing and another test digest of hte sequenced clone.
Next steps are that we are performing the colony PCR again with new primers which means that we are using the iGEM primers which should specifically bind to the constructs which contain the iGEM prefix and suffix add-ons. Since CFP does contain the iGEM restriction sites aswell, but differ in the size of the expected fragment (CD ~1100bp vs. CFP ~700bp) the positive clones should be detectable by the size length.

Cloning of pSB1C3_Affibody_VP2/3

Investigator: Jessica

Comment: Cloning of Affibody and VP2/3 without linker to test if it works without linker

Digestion:

components	pSB1C3_ZEGFR:1907 clone 2 (P268)	pSB1C3_VP2/3 clone 1 (P440)
DNA	14,35	5,2
BSA	2	2
Buffer 4 (10x)	2	2
XbaI	1	1
NgoMIV	-	1
AgeI	1	-
H₂O	-	8,8
Total volume	20,35	20

Comment:Digestion was performed 1,5 hours at 37 °C

Gel:

0,5 g Agarose,50 ml TAE (1%), 5 µl Gelred , at Volt

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

pSB1C3_ZEGFR:1907 clone 2 (P268): c= 2,6ng/µl
pSB1C3_VP2/3 clone 1 (P440: c= 6,7ng/µl

Ligation:

The Ligation was performed as following:

Vector Volume: 5,83µl
Insert Volume: 2,17µl

1 µl T4Ligase buffer (2x)
8 µl (Vector + Insert) mix
1 µl T4Ligase

Incubating for 45 minutes.

Transformation:

Trafo was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with Cm.

PCR of GFP_RFC25 and cloning into pSB1C3_6XHis and pSB1C3_CFP

Investigator: Anissa

Comment:GFP will be cloned into pSB1C3 for biobrick production and upstream of his-tag into into pSB1C3_his for further use

Preparations:
A 1:1000 dilution of the plasmid P416 (pSB1A3_EGFP_HQ clone1). GFP will be extracted and cloned into pSB1C3 iGEM standard plasmid with the RFC25 standard.

PCR protocol:

Ingredients	volume
5X Phusion HF buffer	10 µl
10 mM dNTP mix	1µl
forward primer: O186	2,5 µl
reverse primer: O187	2,5 µl
DNA Template	5 µl
DMSO	0 µl
Phusion Polymerase	0,5 µl
H₂O	28,5 µl
Total volume	50 µl

The following PCR programm was used. Idea is that in the first 8 cycles the primer can only bind to the template added in to the reaction mix. The following 17 cycles afterwards allow the annealing of the complete primer containing prefix and suffix with its restriciton sites.

Cycles	Temperature	Time
	98°C	1
8x	98°C	15"
	54°C	25"
	72°C	24"
17x	98°C	15"
	64°C	25"
	72°C	7"
1x	72°C	5'
Hold 4°C

Digestion of the vectors:

components	pSB1C3_CFP (P51.2)	pSB1C3_6XHis (P300)
DNA	6,6	5,6
BSA	1,5	1,5
Buffer 4 (10x)	1,5	1,5
XbaI	1	1
NgoMIV	-	1
AgeI	1	-
H₂O	3,4	4,4
Total volume	15	15

Comment:Digestion was performed 1,5 hours at 37 °C

Gel:

0,5 g Agarose,50 ml TAE (1%), 3 µl Gelred , at 110 Volt

Comment:The gel showed for the sample p300 no clear band. Perhaps the enzymes did not cut right. The gel-ex-sample of p300 was seperated into an "upper" and a "lower band" sample.

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

pSB1C3 (P51.2): c= 3,95 ng/µl
pSB1C3_6Xhis_upper (P300): c= 8,46 ng/µl
pSB1C3_6Xhis_lower (P300): c= 2,10 ng/µl
PCR GFP_RFC25: c= 6 ng/µl

Digestion of the PCR-product:

components	GFP_RFC25
DNA	30
BSA	4
Buffer 4 (10x)	4
XbaI	1
AgeI	1
H₂O	-
Total volume	40

Comment:Digestion was performed 1,5 hours at 37 °C

Purification of the PCR-product: Was performed accoding to the standard-protocol

Ligation:

The Ligation was performed as following:

P51.2 Volume: 5,83µl
Insert Volume: 2,17µl

P300_upper Volume: 5,83µl
Insert Volume: 2,17µl

P300_lower Volume: 5,83µl
Insert Volume: 2,17µl

1 µl T4Ligase buffer (2x)
8 µl (Vector + Insert) mix
1 µl T4Ligase

Incubating for 30 minutes.

Transformation:

Trafo was performed according to the standard protocol (XL1b). The cells were plated on a agar plates .

PCR for biobrick production: Cloning RepCap into pSB1C3

Investigator: Stefan

Comment:Goal of the experiment was to bring the different RepCap constructs into the pSB1C3 plasmid containing the deletions of the restriction sites interfering with the loop insertions.

Preparations:
Plasmids used:

pAAV_RC_1.2SDMSalI (P158)
pAAV_RC_CapIns_prepSDM (P396)
pAAV_RC_CapIns_noRep (P413)
pAAV_RC_RepCapIns_SDMKpnI (P432)

A 1:1000 dilution of each of the plasmids was prepared.

PCR protocol:

Ingredients	P158 (v/µl)	P396 (v/µl)	P413 (v/µl)	P432 (v/µl)
5X Phusion HF buffer	10	10	10	10
10 mM dNTP mix	1	1	1	1
forward primer: O93	2,5	2,5	2,5	2,5
reverse primer: O120	2,5	2,5	2,5	2,5
DNA Template	2,5	2	3,2	3
DMSO	-	-	-	-
Phusion Polymerase	0,5	0,5	0,5	0,5
H₂O	31	31,5	30,3	30,5
Total volume	50	50	50	50

PCR program:

Comment:For all approaches the following programm was used:

Cycles	Temperature /°C	Time /s
1	98	60
2 (8x step 2-4)	98	15
3	59	25
4	72	70
5 (17x step 5-6)	98	15
6	72	80
71x	72	300
Hold 4°C

Digestion of the vector:

components	pSB1C3_VCK_Bla (P320) (v/µl)
DNA	6,1
BSA	2
Buffer 4 (10x)	2
XbaI	1
AgeI	1
H₂O	7,9
Total volume	20

Comment:Digestion was performed 2 hours at 37 °C.

Gels:
For PCR products:
0,4 g Agarose,50 ml TAE (0,8%), 3 µl Gelred , at 110 Volt; run for ~40 minutes

For vector:
0,5 g Agarose,50 ml TAE (1%), 3 µl Gelred , at 110 Volt; run for ~50 minutes

Freiburg10 Biobrick vector digest of RepCap.jpg

Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the vector:

pSB1C3 (P320): c= 11,97 ng/µl

Digestion of the PCR-products:

Comment:Digestion was performed for all PCR products alike.

components	PCR products (v/µl)
DNA	30
BSA	4
Buffer 4 (10x)	4
XbaI	1
AgeI	1
H₂O	-
Total volume	40

Comment:Digestion was performed 2 hours at 37 °C.

PCR purification:
Purification was performed accoding to the standard protocol. The following concentrations were measured:

PCR of P158: c= 7,68 ng/µl
PCR of P396: c= 17,65 ng/µl
PCR of P413: c= 24,17 ng/µl
PCR of P432: c= 18,23 ng/µl

Ligation:
1 µl T4 Ligase buffer (2x) 8 µl DNA mix 1 µl T4 Ligase
Incubating for 45 minutes at room temperature.

The Ligation was performed using the following amounts of DNA:

Vector Volume: 1,98 µl
P158 Volume: 6,02 µl

Vector Volume: 3,45 µl
P396 Volume: 4,55 µl

Vector Volume: 4,07 µl
P413 Volume: 3,93 µl

Vector Volume: 3,51 µl
P413 Volume: 4,49 µl

Transformation:

Trafo was performed according to the standard protocol using XL1b cells. The cells were plated on a agar plates containing chlorampenicol.

Mini-Preps

Investigator: Hanna

Mini-Preps were performed according to standard protocol:
pSB1C3_ITR_phTERT_b-globin__mGMK clone 1 (P483)
pSB1C3_ITR_phTERT_b-globin__mGMK clone 2 (P484)
pSB1C3_lITR_phTERT_b-globin__mVenus clone 1 (P485)
pSB1C3_lITR_phTERT_b-globin__mVenus clone 2 (P486)
pSB1C3_lITR_CMV_b-globin__mGMK clone 1 (P487)
pSB1C3_lITR_CMV_b-globin__mGMK clone 2 (P488)
pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413) clone 1 (P489)
pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413) clone 2 (P490)
pSB1C3_VP123 (aus pAAV_RC_1.2 SDM SalI P158) clone 1 (P491)
pSB1C3_VP123 aus (pAAV_RC_1.2 SDM SalI P158) clone 2 (P492)

Colony PCR of pCerulean_CFP_MiddleLinker, pCerulean_6xHis_MiddleLinker and pCerulean_"Linker"_Zegfr:1907 constructs (Linker library)

Investigator: Hanna

Comment: Because we found out that there's also a SacI restriction site in the VP2/3 sequence - due to the PstI (4073) mutagenesis - the first try of cloning VP2/3 to the C-terminus of the Zegfr:1907, CFP and His-Tag constructs was discarded.
Fortunately Anissa already repeated this experiment yesterday. Because we're not sure whether cloning was successful (one plate was without any colonies and has to be done one more time), we decided to perform a colony PCR before continuation with (at least 22!) mini-preps. We chose primer which bind in the VP2/3 region and therefore amplify a VP2/3 fragment - given, that VP2/3 was successfully cloned downstream of the linker. One further advantage is, that we can also save the test digestions.
A Mastermix was prepared for 43 samples:

5x Crimson Taq Mg-free reaction buffer: 215 µL
10 mM dNTP: 21.5 µL
10 µM Forward primer (Primer Cap 3500 for): 21.5 µL
10 µM Reverse Primer (Primer Cap 4200 rev): 21.5 µL
Crimson Taq DNA Polymerase: 5.375 L
Nuclease-Free water: 790.125 µL

Total: 1075 µL -> 25 µL reactions

2-4 colonies were picked with a tooth pick from each plate (11). 25 µL master mix and 5 ml LB + Kanamycin were inocculated.

PCR program:
According to standard protocol provided by Gerrit

95°C, 30
95°C 25
50.5°C, 40
68°C, 60 --> repeat 2-4 27 times
68°C, 300
4°C, hold

Tomorrow morning the PCR products will be tested on an analytic gel. If the samples will be positive, they will be mini-preped - if not cloning will be done again.
Tod do: Cloning of VP2/3 into pCerulean_CFP_MiddleLinker.

Sequencing analysis of VP1-ko and VP2-ko in severeal pAAV vectors

Investigator: Stefan
Primer used (for all plasmids): 2800 rev (O38)

Sequenced plasmid:

pAAV_RC_1.2SDMSalI_VP1-ko clone 1 (P449)
pAAV_RC_CapIns_prepSDM_VP1-koclone 1 (P451)
pAAV_RC_CapIns_noRep_VP1-ko clone 1 (P453)
pAAV_RC_RepCapIns_SDMKpnI_VP1-ko clone 1 (P455)
pAAV_RC_1.2SDMSalI_VP2-ko clone 1 (P457)
pAAV_RC_CapIns_prepSDM_VP2-ko clone 1 (P459)
pAAV_RC_CapIns_noRep_VP2-ko clone 1 (P461)
pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 1 (P463)

Comment:All sequences showed that the site directed mutagenesis worked out for all constructs. All sequencing results revealed problems in the area shown below:

Freiburg10 Sequencing VP1 2 ko problem(2).jpg

Freiburg10 Sequencing VP1 2 ko problem(1).jpg

122. labday 16.09.2010

Trafo of T4 ligation, Results of Quickligation

Investigator: Achim

No clones on the Quickligation plates. Trafo of T4 Ligation was carried out in XL1B cells. Results will be evaluated tomorrow.

Mini Prep and test digestion of several constructs

Investigator: Anissa, Bea

Comment: Several test digestions have been performed for verifying the cloned fragment into the plasmid backbone. The constructs for reassembling the vector plasmid (pSB1C3_leftITR_PROMOTER_betaglobin_GOI) were digested with EcoRI and NgomIV, the SV40 construct was digested with EcoRI and PstI.

Test digestion of the constructs:

pSB1C3_leftITR_phTERT_betaglobin_mVenus (P485-486)
pSB1C3_leftITR_phTERT_betaglobin_mGMK (P483-484)
pSB1C3_leftITR_pCMV_betaglobin_mGMK (P487-488)
pSB1C3_SV40 (P493-494)

Expected size fragments are:

(P485-486): 1150bp, 2780bp
(P483-484): 1150bp, 2650bp
(P487-488): 1300bp, 2600bp
(P493-494): 270bp, 2030bp

Results: Test digestion of the constructs looks well, except for the construct 494 which is one clone of the construct pSB1C3_SV40. Since the other clone is fine, the construct was discarded. The control K437 is the plasmid pSB1C3_leftITR_hTERT_beta-globin without the assembled GOI (mGMK and mVenus repsectively). The GOI´s are in the RFC25 standard therefore leading to scar with a additional NgoMIV after fusing it to the before mentioned construct which is in the RFC10 standard. Therefore, test digestion was carried out with NgoMIV and EcoRI leading to fragments which can be separated from the backbone in a 1% agarose gel. The control vector should only be linearized because it does not contain any NgomIV rstriction site.

Freiburg10 pSB1C3 leftITR Promoter betaglob GOI 16 09 2010.jpg

Test digestion of pSB1C3_VP123

Investigator: Jessica

Samples	Plasmid	Clone	Concentration [ng/µl]
P489	pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413)	1	350
P490	pSB1C3_VP123 (aus pAAV_RC_CapIns_noRep P413)	2	395,5
P491	pSB1C3_VP123 (aus pAAV_RC_1.2 SDM SalI P158)	1	403
P492	pSB1C3_VP123 (aus pAAV_RC_1.2 SDM SalI P158)	2	411

Test digestion:

components	Volume for each sample /µl
DNA	2,6/2,3/2,3/2,2
BSA (10x)	1
Buffer 4 (10x)	1
Enzyme NgoMIV	0,5
Enzyme SapI	0,5
H2O	4,4/4,7/4,7/4,8
Total volume /µl	10

Preparation of gel:

Incubation time: 1,5 h, Incubation temperature: 37°
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at Volt, running time: minutes

Expected fragment sizes:

3244 bp

767 bp

Comment: Sample P489 was sent for sequencing with primer VR2. Sequencing result looks good, VP123 is in

Continuation of preparation of competent E.coli

Investigator: Jessica
preparation of competent BL21 was finished according to the standard protocol

aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer

don't use before results of testtrafo is writen down
testtrafo was made with pUC and Amp

Mini prep of CD-clones, which were identified by colony pcr

Investigator: Kira

c(clone1) = 160,29 ng/ul
c(clone2) = 167,98 ng/ul
c(cloone3) = 181, 51 ng/ul

clone 3 was sent for sequencing

Repetition: PCR for biobrick production of P40 promotor

Investigator: Stefan

Comment: During trouble-shooting we realized that the reverse primer P40_rev (O153) was designed forward and therefore does not work. A new primer will have to be designed and ordered to repeat the PCR.

Results of Colony PCR

Investigator: Hanna

Comment: In order to find out, whether the VP2/3 or the VP2/3_Capins fragment was successfully cloned into pCerulean_Zegfr:1907_"Linker" (--> Linker library), pCerulean_6xHis_MiddleLinker and pCerulean_CFP_MiddleLinker, a colony PCR was performed yesterday. From each plate 2-4 clones were picked, so that we got 41 samples (including one positive control = pAAV_RC). With VP_3500_for and VP_4200_rev primer a 880 fragment should be amplified of each colony, if cloning functioned.

Samples were loaded onto a 1% agarose gel:

1. pCerulean_Zegfr:1907_ShortLinker_VP2/3
2. pCerulean_Zegfr:1907_MiddleLinker_VP2/3_Capins
3. pCerulean_Zegfr:1907_LongLinker_VP2/3
4. pCerulean_6xHis_MiddleLinker_VP2/3
5. pCerulean_Zegfr:1907_SEG_VP2/3
6. pCerulean_6xHis_MiddleLinker_VP2/3_Capins
7. pCerulean_CFP_MiddleLinker_VP2/3_Capins
8. pCerulean_Zegfr:1907_ShortLinker_VP2/3_Capins
9. pCerulean_ Zegfr:1907_SEG_VP2/3_Capins
10. pCerulean_Zegfr:1907_LongLinker_VP2/3_Capins
11. pCerulean_Zegfr:1907_MiddleLinker_VP2/3

As can be seen in the gel picture, it seemed that just clone 9.3, 10.2, 11.1 and 11.2 were VP2/3 positive. Curiously the positive control (K) was negative.

These 4 clones were mini-preped by Achim and sent for sequencing to GATC: primer = GATC_std_CMV-F.

Because I found out that some things went wrong during VP cloning due to false geneious annotations and theoretical cloning approaches, all pAAV_RC constructs (with/without Cap or Rep-insert, 4-fold mutated, PvuII insertion, KpnI-backmutation,...) were theoretically prepared again (see geneious folder: "pAAV_RC constructs (DO NOT CHANGE!!!)").
In addition to that the theoretical cloning of the final N-terminal fusion approaches to VP2/3 were performed once again with these correct sequences. With the help of Sven cloning of the not yet positive approaches were repeated (The ones which were negative in colony PCR).

To do (tomorrow): Pciking clones, analyze sequencing. Check and plan HSPG-KO approaches and Zegfr:1907

Practical Cloning:

- Vector: pCerulean_CFP_MiddleLinker (P407), pCerulean_Zegfr:1907_MiddleLinker (P408), pCerulean_Zegfr:1907_SEG (P409), pCerulean_Zegfr:1907_ShortLinker (P410), pCerulean_Zegfr:1907_LongLinker (P412), pCerulean_6xHis_MiddleLinker (P374)
- Insert: pSB1C3_VP2/3 (P440), pSB1C3_VP2/3_ins_cap (P444)
- new vector name: pCerulean_CFP_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_SEG_VP2/3, pCerulean_Zegfr:1907_ShortLinker_VP2/3, pCerulean_Zegfr:1907_LongLinker_VP2/3, pCerulean_6xHis_MiddleLinker_VP2/3 AND pCerulean_CFP_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_SEG_VP2/3-insCap, pCerulean_Zegfr:1907_ShortLinker_VP2/3-insCap, pCerulean_Zegfr:1907_LongLinker_VP2/3-insCap, pCerulean_6xHis_MiddleLinker_VP2/3-insCap
buffer used:4

Comment Because the VP2/3 frament has approximately the same size as the backbone pSB1C3 it may be difficult to separate them on the agarose gel. Therefore a single cutter enzyme (MscI) was chosen to digests the backbone. This enzyme also works in buffer 4 + BSA and needs to be incubated at 37°C.

- Vector: AgeI, SpeI
- Insert: NgoMIV, SpeI, SacI

Digestion

Incubation: 2 h

Agarose gel

Green boxes were cut out.

Gelex, ligation and trafo were performed according to standard protocol.
Next step: Colony PCR of each ligation approach.

122. labday 17.09.2010

Colony PCR of N-terminal fusion approach

Investigator: Achim

Comment:

Sequencing analysis of pSB1C3_VP123

Investigator: Bea

Comment: The constrcuct was created by performing a PCR with the pAAV_RC_inscap construct. The sequence analysis reveals that the suffix looks well. Additionally the mutation from TAA to TGA can be seen.

Freiburg10 Seqanalysis pSB1C3 VP123 inscap.jpg

ViralBricks: Picking clones from T4 ligation

Investigator: Achim

Sequencing results N-terminal fusion - part 1

Investigator: Hanna

Comment: Colony PCR delivered that 4 clones contained the VP2/3 or VP2/3_Capins sequence. These clones were sent for sequencing:

1. pCerulean_Zegfr:1907_LongLinker__VP2/3_Capins:

Sequencing looked well: VP2/3_Capins was successfully fused to the C-terminus of the Long-Linker. The additional basepair at the beginning of the Affibody was misinterpreted (as one can see in the chromatogram) by Geneious.

2. pCerulean_Zegfr:1907_MiddleLinker__VP2/3 clone 1:

Sequencing looked well: VP2/3 was successfully fused to the C-terminus of the Middle-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious.

3. pCerulean_Zegfr:1907_MiddleLinker__VP2/3 clone 2:

Sequencing looked well: VP2/3 was successfully fused to the C-terminus of the Middle-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious.

4. pCerulean_Zegfr:1907_SEG__VP2/3_Capins:

Sequencing looked well: VP2/3_Capins was successfully fused to the C-terminus of the SEG-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious.

Mini-Prep and test digestion of several constructs

Investigator: Stefan

Glycerol stocks were prepared:

B430 = pSB1C3_001_RC_4fold mutated clone 1
B431 = pSB1C3_001_RC_4fold mutated clone 2
B432 = psB1C3_001_RC_InsRepCap clone 1
B433 = psB1C3_001_RC_InsRepCap clone 2
B434 = pSB1C3_001_RC_InsCap clone 1
B435 = pSB1C3_001_RC_InsCap clone 2
B436 = pSB1C3_001_RC_InsRepCap_KpnIback clone 1
B437 = pSB1C3_001_RC_InsRepCap_KpnIback clone 2
B438 = pSB1C3_001_VP2/3insCap clone 1
B439 = pSB1C3_001_VP2/3insCap clone 2
B440 = pSB1C3_ZEGFR:1907_VP2/3 clone 1
B441 = pSB1C3_ZEGFR:1907_VP2/3 clone 2
B442 = pSB1C3_GFP_RFC25_upper band clone 1
B443 = pSB1C3_GFP_RFC25_upper band clone 2
B444 = pSB1C3_GFP_RFC25_lower band clone 1
B445 = pSB1C3_GFP_RFC25_lower band clone 2
B446 = pSB1C3_GFP_RFC25 clone 1
B447 = pSB1C3_GFP_RFC25 clone 2

Mini-Prep was performed according to the standard protocol

P506 pSB1C3_001_RC_4fold mutated clone 1
P507 pSB1C3_001_RC_4fold mutated clone 2
P508 psB1C3_001_RC_InsRepCap clone 1
P509 psB1C3_001_RC_InsRepCap clone 2
P510 pSB1C3_001_RC_InsCap clone 1
P511 pSB1C3_001_RC_InsCap clone 2
P512 pSB1C3_001_RC_InsRepCap_KpnIback clone 1
P513 pSB1C3_001_RC_InsRepCap_KpnIback clone 2
P514 pSB1C3_001_VP2/3insCap clone 1
P515 pSB1C3_001_VP2/3insCap clone 2
P516 pSB1C3_ZEGFR:1907_VP2/3 clone 1
P517 pSB1C3_ZEGFR:1907_VP2/3 clone 2
P518 pSB1C3_GFP_RFC25_upper band clone 1
P519 pSB1C3_GFP_RFC25_upper band clone 2
P520 pSB1C3_GFP_RFC25_lower band clone 1
P521 pSB1C3_GFP_RFC25_lower band clone 2
P522 pSB1C3_GFP_RFC25 clone 1
P523 pSB1C3_GFP_RFC25 clone 2
P524 pSB1C3_VP2/3
P525 pSB1C3_VP2/3_ins_cap

Test digestion:
Test digestion was performed for plasmids P506, P507, P510 to P513 and for P522 and P523. Test digestion of P514 to P521 will be performed tomorrow. Preps for P524 and P525 were inocculated from glycerol stock and therefor no digestion will be performed. The constructs were digested using XbaI and AgeI for 40 minutes at 37°C.

components	P470-P481	P470-P481
DNA	2	2
Buffer 4 (10x)	1	1
XbaI	0,4	0,4
AgeI	0,4	0,4
H₂O	7,2	6,2
Total volume	10	10

Preparation of gel:

0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 40 minutes

Freiburg10 test digestion pSB1C3 001 RepCaps pSB1C3 GFP RFC25.jpg

Comment:

Sent for sequencing:
Primer used:
VR2
plasmid no:
P522
tube labeled: SB_1

Sent for sequencing

Investigator: Stefan
pSB1C3_SV40 (P493) was sent for sequencing using VR2 primer. Results have to be checked tomorrow.

Sequencing analysis of VP2/3 insertion into pCerulean_VP1up_NLS_targeting motif

Investigator: Stefan
Comment: Sequences can be found in GATC Watchbox (date 15.10.10) labeled SB_1 (VP2/3 insertion) and SB_2 (VP2/3_insCap insertion).

Freiburg10 Sequencing VP2 3 insertion.JPG

Freiburg10 Sequencing VP2 3 insCap insertion.JPG

Biobrick production of VP2/3- and VP2/3 cap-Gsat_linker

Motivation: Production of fusion proteins

Investigator: Kira

Digestions of backbones and inserts:

Components	VP2/3	VP2/3 cap	Gsat linker
DNA	7	7	7
BSA (100x)	0	0	0
Buffer no. 4 (10x)	2,5	2,5	2,5
EcoRI	0,5	0,5	0,5
NgoMIV	0,5	0,5	0
XbaI	0	0	1,25
H₂O	14,5	14,5	13,75
Total volume	25	25	25

incubation @ 37 C for approx. 3 h

1% agarose gel

Ligation:

T4 ligase was used. ligation over night @ 18 C

VP2/3-mix 8 ul (3,7 ul vector + 4,3 ul insert)
VP2/3 cap-mix 8 ul (3,7 ul vector + 4,3 ul insert)

123. labday 18.09.2010

Continuation of preparation of competent E.coli

Investigator: Jessica
preparation of competent XL1blue was finished according to the standard protocol

aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer

don't use before results of testtrafo is writen down

Test digestion of pSB1C3_ZEGFR:1907_VP2/3

Investigator: Jessica

Samples	Plasmid	Clone	Concentration [ng/µl]
P514	pSB1C3_001_VP2/3insCap	1	318.9
P515	pSB1C3_001_VP2/3insCap	2	321.8
P516	pSB1C3_ZEGFR:1907_VP2/3	1	191,6
P517	pSB1C3_ZEGFR:1907_VP2/3	2	210,8
P518	pSB1C3_GFP_RFC25_upper band	1	138.3
P519	pSB1C3_GFP_RFC25_upper band	2	72.3
P520	pSB1C3_GFP_RFC25_lower band	1	141.6
P521	pSB1C3_GFP_RFC25_lower band	2	127.5

Test digestion:

components	Volume for each sample P516/517 /µl	Volume for each sample P514/515 /µl	Volume for each sample P518-521 /µl
DNA	2,5	3,5	1,5
BSA (10x)	1	1	1
Buffer 1/4/4 (10x)	1	1	1
Enzyme NgoMIV	0,5	-	-
Enzyme DraIII	0,5	-	-
Enzyme XbaI	-	-	0,4
Enzyme AgeI	-	-	0,4
Enzyme MscI	-	0,4	-
Enzyme SpeI	-	0,4	-
H2O	4,5	3,7	5,7
Total volume /µl	10	10	10

Preparation of gel:

Incubation time: 1,5 h, Incubation temperature: 37°
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt

Expected fragment sizes:

P516/517: 3540bp / 651bp
P514/515: 1300bp / 2700bp
P518-521: 2000bp / 700bp

Comment: All samples look OK! Samples will be sent for sequencing on monday.

Mini-Prep of pSB1C3_SV40 clone 1 (B420)

Investigator: Jessica
was done because of the low concentration of the plasmid P493

P526: pSB1C3_SV40 clone 1 (=B420) c= 176,0 ng/µl

New aliquots (60µl) of BL21 can be used ! (Jessica)

New Colony PCR of N-terminal fusion approach

Investigator: Hanna

Comment: Because just 3 clones of the colony PCR (yesterday) were VP2/3 positive, new colonies were picked for a colony PCR. We assumed that we can find some positive clones, because the 3 successful ligations contained VP2/3 AND VP2/3_Capins, so that we concluded, that digestion worked and that we just have to search for positive clones on the plates.

A mastermix for 28 samples was prepared:

10x Taq Buffer: 28 µL
MgCl₂ (50 mM): 2.8 µL
dNTPs: 5.6 µL
Primer 1 (4200 rev = O42): 5.6 µL
Primer 2 (3500 for = O39): 5.6 µL
Taq polymerase: 1.4 µL
H₂O: 231 µL

--> Volume for each reaction: 10 µL

4 colonies were picked with a tooth-pick from each plate (6), inoculated into the 10 µL mastermix and then put into 5 µL DYT containing Kanamycin.
As positive control 1 ng of P158 = pAAV_RC_SDM_SalI and as negative control 1 ng of P42 = pAAV_RFC25 were used.

PCR program:

95°C, 6'

30x

95°C, 25
50.5°C, 30
68°C, 54

1x

68°C, 5'

Hold 4°C

Gel picture:

All PCR reactions delivered positive VP2/3 results (expected size: 880 bp). Unfortunately also the negative control was positive. We decided that 2 clones of each plate will be mini-preped. The remaining 2 clones will be pelletized and stored at -20°C. On monday several test digestions will be performed (perhaps 1 of each plate).

Trafo of pSB1C3_GSAT_VP2/3 and pSB1C3_GSAT_VP2/3_Capins

Investigator: Hanna

Comment: Because we just used the Long-, Middle-, Short- and SEG-Linker for the small linker library, we decided also to perform the N-terminal fusion to VP2/3 with the GSAT linker.
Yesterday, VP2/3 and VP2/3_Capins were cloned to the C-terminus of the GSAT linker. Ligation was performed over night. New plasmids: pSB1C3_GSAT_VP2/3 and pSB1C3_GSAT_VP2/3_Capins
Trafo was performed according to standard protocol: XL1b cells, 2 µL DNA. Plates will be stored over night @ 37°C room.
To do tomorrow: Picking clones.

Colony-PCR of pSB1C3_CD

Investigator: Bea

Comment: Since the colony PCR of the last try did not work exactly the way we expected it, another colony PCR approach with new primers was conducted.

The protocol:

Forward primer used: iGEM RFC25
Reverse primer used: iGEM RFC25
Cells: XL1-Blue

A mastermix for 37 samples was prepared for using it in this approach and the colony PCR for the loop insertion motif Z34C.

10x Taq Buffer: 1.0 µL
MgCl₂ (50 mM): 0.1 µL
dNTPs: 0.2 µL
Primer iGEM RFC25 forward: 0.2 µL
Primer iGEM RFC25 reverse: 0.2 µL
Taq polymerase: 1.4 µL
H₂O: 231 µL
Total volume for each reaction: 10 µL

PCR programm used:

Corresponding inoculated cells put in the 37°C room over night at 21:00.

BioBrick Assembly of ITR_phTERT_betaglobin_mVenus to hGH_rightITR

Investigator: Bea

Comment: For assembling the last BioBricks together in order to obtain the fully assembled vectorplasmid which contains the tumorspecific promoter phTERT and the GOI mVenus.

Digestion of the constructs:

P485 = pSB1C3_leftITR_phTERT_betaglobin_mVenus c=257 ng/µL
P440 = pSB1C3_hGH_rightITR c=105 ng/µL

Components	v_P485 /µL	v_P186/µL
DNA	4	14
BSA (10x)	2	2.5
Buffer no. 4 (10x)	2	2.5
EcoRI	1	1
AgeI	1	1
H₂O	10	4
Total volume	20	25

The expected fragments of the digested constructs were: The underlined fragments represent the fragments which correspond to the desired construct which can be used for ligation.

P485: 3911bp
P186: 657, 2072bp

Loading plan:

M    P485    P186

Results:

Freiburg10 pSB1C3 phTERT mVenus 18 09 2010.jpg

After gel extraction has been performed, the ligation was carried out.

Ligation:

v_=P485=4,96µL
v_=P309=3,04µL

The ligation mix was transformed into XL1-Blue cells and plated on agar plates containing chloramphenicol.

Next steps:
Picking clones and perform Mini-Prep. After the Mini-Preps have been performed this construct needs to be sequenced and tested in cell culture.

Colony-PCR of ViralBricks

Investigator: Achim

Comment: We decided to screen the new ViralBrick clones for the right insert via colony-pcr. This way we avoid unnecessary minipreps of clones containing religated vector or BLA.

The protocol:

Forward primer used: iGEM RFC25
Reverse primer used: iGEM RFC25
Cells: XL1-Blue

A mastermix for 37 samples was prepared for using it in this approach and the colony PCR for the loop insertion motif Z34C.

10x Taq Buffer: 1.0 µL
MgCl₂ (50 mM): 0.1 µL
dNTPs: 0.2 µL
Primer iGEM RFC25 forward: 0.2 µL
Primer iGEM RFC25 reverse: 0.2 µL
Taq polymerase: 1.4 µL
H₂O: 231 µL
Total volume for each reaction: 10 µL

PCR programm used:

Corresponding inoculated cells put in the 37°C room over night at 21:00.

=> Go to Labjournal September part 2 (labday 124 - 135)

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 <ul>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March (labday 1)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April (labday 2 - 5)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May (labday 6 - 17)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June">June</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June">June (labday 18 - 45)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July">July</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July">July (labday 46 - 75)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August part 1 (labday 76 - 92)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2">August part 2 (labday 93 - 106)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
 </ul>
+</div>
 </html>
 ==September==
@@ Line 252: / Line 147: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning of Z<sub>EGFR</sub>:1907_Middle-Linker,Z<sub>EGFR</sub>:1907_Short-Linker, Z<sub>EGFR</sub>:1907_SEG-Linker and Z<sub>EGFR</sub>:1907_Long-Linker into pCerulean</b></p>====
 <b>Investigator: Stefan </b>
-<p style="font-size:13px; color:red;"><b>Comment</b>:</p>
 <br/>
 '''Digestion:'''
@@ Line 281: / Line 175: @@
 <br />
 <br />
-[[Image:Freiburg10 pCerulean affi linker.jpg|500px|]]
+[[Image:Freiburg10 pCerulean affi linker.jpg|thumb|center||500px|]]
 <br />
 <br />
@@ Line 315: / Line 209: @@
 Trafo was performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol
 <br>
 ===<p style="font-size:17px; background-color:#00dd77;">108. labday 02.09.2010</p>===
@@ Line 562: / Line 454: @@
 ===<p style="font-size:17px; background-color:#00dd77;">109. labday 03.09.2010</p>===
-====<p style="font-size:15px; background-color:#ff00ff;"><b>Miniprep and test digestion of pSB1C3_lITR_pTERT_ßglobin_mVenus_hGH_rITR</b></p>====
+====<p style="font-size:15px; background-color:#66bbff;"><b>Miniprep and test digestion of pSB1C3_lITR_pTERT_ßglobin_mVenus_hGH_rITR</b></p>====
 <b>Investigator: Achim <br /></b>
 <br/>
@@ Line 570: / Line 462: @@
 <br/>
-====<p style="font-size:15px; background-color:#ff00ff;"><b>Sequencing results</b></p>====
+====<p style="font-size:15px; background-color:#66bbff;"><b>Sequencing results</b></p>====
 <b>Investigator: Hanna <br /></b>
 <br/>
@@ Line 585: / Line 477: @@
 <br/>
-====<p style="font-size:15px; background-color:#ff00ff;"><b>Cloning of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker</b></p>====
+====<p style="font-size:15px; background-color:#66bbff;"><b>>Cloning of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker</b></p>====
 <b>Investigator: Hanna <br /></b>
 <br/>
@@ Line 781: / Line 673: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)</b></p>====
-Investigator: Patrick
+'''Investigator: Patrick'''
 <br />
@@ Line 795: / Line 687: @@
 <br>
-see also http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Cloning_CFP_middlelinker_.28from_pSB1C3_CFP_middlelinker.2C_P276.29_into_pCerulean_.28P273.29
+[[Image:Mistake.png|thumb|right|200px| ;-) bitte ausfüllen]]
-[[Image:Mistake.png|thumb|right|400px| ;-) bitte ausfüllen]]
+see also:
+http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Cloning_CFP_middlelinker_.28from_pSB1C3_CFP_middlelinker.2C_P276.29_into_pCerulean_.28P273.29
+<br/>
 Testdigestion: 7 µl DNA sample, 1 µl Buffer 4 (10x), 1 µl BSA, 0,5 µl Xba, 0,5 µl PstI-HF
 <br>
@@ Line 1,104: / Line 998: @@
 <br />
 <br />
-[[Image:Freiburg10 pCerulean pMA pAAV.png|500px|]]
+[[Image:Freiburg10 pCerulean pMA pAAV.png|thumb|center||600px|]]
 <br />
 <br />
@@ Line 1,139: / Line 1,033: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273): Sequencing results of P381 and P385</b></p>====
-Investigator:Patrick
+'''Investigator:Patrick'''
 Sequencing results of P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev:
@@ Line 1,208: / Line 1,102: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition: Prepatation for SDM: cloning of Cap into pAAV</b></p>====
-Investigator: Stefan
+'''Investigator: Stefan'''
 [[Image:Freiburg10_Kopfkratzen.gif|thumb|right|Schönen Abend dir noch]]
@@ Line 1,285: / Line 1,179: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Preparation of PBS Buffer</b></p>====
-Investigator: Volker
+'''Investigator: Volker'''
 Two liters of PBS buffer were prepared for the column purification of the viral particles. The following protocol was used:
@@ Line 1,306: / Line 1,200: @@
 Mini-Prep was performed according to the standard protocol. Two preps were prepared:
 <br />
-<li>P363 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (1) = 215,6 ng/µl
+<ul>
-<li>P364 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (2) = 230,5 ng/µl
+<li>P363 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (1) = 215,6 ng/µl</li>
+<li>P364 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (2) = 230,5 ng/µl</li>
+</ul>
 <br />
-====<p style="font-size:15px; background-color:#ff00ff;"><b>Picking clones of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker</b></p>====
+====<p style="font-size:15px; background-color:#66bbff;"><b>>Picking clones of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker</b></p>====
 <b>Investigator: Hanna<br /></b>
 <br/>
@@ Line 1,480: / Line 1,376: @@
 Mini-Prep was performed according to the standard protocol
 <br />
-<li>P394 = pAAV_RC_CapIns_prepSDM clone 1 = 296,42 ng/µl
+<ul>
-<li>P395 = pAAV_RC_CapIns_prepSDM clone 2 = 274,92 ng/µl
+<li>P394 = pAAV_RC_CapIns_prepSDM clone 1 = 296,42 ng/µl</li>
-<li>P396 = pAAV_RC_CapIns_prepSDM clone 3 = 498,39 ng/µl
+<li>P395 = pAAV_RC_CapIns_prepSDM clone 2 = 274,92 ng/µl</li>
-<li>P397 = pAAV_RC_CapIns_prepSDM clone 4 = 487,67 ng/µl
+<li>P396 = pAAV_RC_CapIns_prepSDM clone 3 = 498,39 ng/µl</li>
-<li>P398 = pAAV_RC_CapIns_prepSDM clone 5 = 469,96 ng/µl
+<li>P397 = pAAV_RC_CapIns_prepSDM clone 4 = 487,67 ng/µl</li>
-<li>P399 = pAAV_RC_CapIns_prepSDM clone 6 = 517,06 ng/µl
+<li>P398 = pAAV_RC_CapIns_prepSDM clone 5 = 469,96 ng/µl</li>
-<li>P400 = pAAV_RC_CapIns_prepSDM clone 7 = 451,68 ng/µl
+<li>P399 = pAAV_RC_CapIns_prepSDM clone 6 = 517,06 ng/µl</li>
-<li>P401 = pAAV_RC_CapIns_prepSDM clone 8 = 479,18 ng/µl
+<li>P400 = pAAV_RC_CapIns_prepSDM clone 7 = 451,68 ng/µl</li>
+<li>P401 = pAAV_RC_CapIns_prepSDM clone 8 = 479,18 ng/µl</li>
+</ul>
 <br />
 Constructs were digested with XcmI and Acc65I for 70 minutes at 37°C
@@ Line 1,510: / Line 1,408: @@
 <br />
 <br />
-[[Image:Freiburg10 pAAV RC CapIns prepSDM.png|500px]]<br />
+[[Image:Freiburg10 pAAV RC CapIns prepSDM.png|thumb|center||700px]]<br />
 <br />
 <br />
@@ Line 1,527: / Line 1,425: @@
 </ul>
 pAAV_RC_inserts clone1 (P326) was sent for sequencing using the following primers:
-* VP1 primer for pKex rev
+<ul>
-* SK
+<li>VP1 primer for pKex rev</li>
-* Primer Cap 2800 for
+<li>SK</li>
-* Primer Cap 2800 rev
+<li>Primer Cap 2800 for</li>
-* Primer Cap 3500 for
+<li>Primer Cap 2800 rev</li>
+<li>Primer Cap 3500 for</li>
+</ul>
 ===<p style="font-size:17px; background-color:#00dd77;">113. labday 07.09.2010</p>===
@@ Line 1,540: / Line 1,440: @@
 *Sequencing confirmed the 453 BAP ligation contains the correct sequence. 453 Z34C and 587 Z34C didn't contain inserts, 587 KO Z34C still contained the BLA placeholder.
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition of CD biobrick production</b></p>====
+<b>Investigator: Kira</b><br>
 Comment: Despite the results of the test digestion, the samples, which were sent for sequencing do not contain the GOI. Thus the whole procedure will be repeated today.
-<b>Investigator: Kira</b><br>
 DNA sample was diluted 1:100
 <br>
 {| border="1"
@@ Line 1,580: / Line 1,479: @@
 |||60°C||25"
 |-
-|||72°C||60"
+|||72°C||40"
 |-
 |17x||98°C||15"
@@ Line 1,586: / Line 1,485: @@
 |||65°C||25"
 |-
-|||72°C||60"
+|||72°C||40"
 |-
 |1x||72°C||5'
@@ Line 1,621: / Line 1,520: @@
 % agarose gel <br />
-[[Image:Freiburg10 PCR product and digested pSB1C3 vector.jpg|500px|]]
+[[Image:Freiburg10 CD_2010_09_07.jpg|500px|]]
 Digestion of PCR product:
@@ Line 1,776: / Line 1,675: @@
 <br />
 <br />
-[[Image:Freiburg10 4x mutant Cap.jpg|500px|]]
+[[Image:Freiburg10 4x mutant Cap.jpg|thumb|center||550px|]]
 <br />
 <br />
@@ Line 1,843: / Line 1,742: @@
 <br />
-<p style="font-size:13px; color:#003399;"><b>To do</b>: A new approach of the ViralBricks production has to be done tomorrow. A possible reason for the failure of the reactions is that the restriction enzymes didn't cut in the kenow buffer.  </p>
+<p style="font-size:13px; color:#003399;"><b>To do</b>: A new approach of the ViralBricks production has to be done tomorrow. A possible reason for the failure of the reactions is that the restriction enzymes didn't cut in the klenow buffer.  </p>
 ====<p style="font-size:15px; background-color:#66bbff;">'''Preparation for Midi-Preps of pAAV_mVenus, pHelper and pAAV_RC'''</p>====
-Investigator: Kerstin
+'''Investigator: Kerstin'''
 <ul><li>25 ml DYT was prepared with 25 µl Ampicillin and inoculated with glycerol stocks of pAAV_mVenus, pHelper and RC, each were incubated over night in 37°C room.</li>
@@ Line 1,856: / Line 1,755: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Sequencing</b></p>====
-Investigator: Hanna
+'''Investigator: Hanna'''
 <br/>
 pCerulean_Zegfr:1907_ShortLinker, pCerulean_Zegfr:1907_MiddleLinker, pCerulean_Zegfr:1907_LongLinker, pCerulean_Zegfr:1907_SEG and pCerulean_CFP_MiddleLinker were sent for sequencing.<br/>
@@ Line 2,053: / Line 1,952: @@
 | '''Ingredients''' || align="right" |Volume / µl
 |-
-| 5X Phusion HF buffer ||  align="right" |10
+| 5x Phusion HF buffer ||  align="right" |10
 |-
 | 10 mM dNTP mix||  align="right" |1
@@ Line 2,080: / Line 1,979: @@
 |||98||60
 |-
-|8x||98||15
+|8x (step 2 - 4)||98||15
 |-
 |||61||25
@@ Line 2,086: / Line 1,985: @@
 |||72||3
 |-
-|17x||98||15
+|17x (step 5- 7||98||15
 |-
 |||69||25
@@ Line 2,113: / Line 2,012: @@
 | align="left" | XbaI ||align="left"| 1
 |-
-| align="left" | PstI ||align="left"| 4
+| align="left" | PstI ||align="left"| 1
 |-
 | align="left" | H<sub>2</sub>O ||align="left"| 4
@@ Line 2,154: / Line 2,053: @@
 <b>PCR progam: </b>
-primers used:<br/>
+The following primers were used:<br/>
-<li>CMV_forward_qPCR<br/>
+<ul>
-<li>CMV_reverse_qPCR<br/>
+<li>CMV_forward_qPCR</li>
+<li>CMV_reverse_qPCR</li>
+</ul>
 <br>
 {| border="1"
@@ Line 2,189: / Line 2,089: @@
 |||95||600
 |-
-|35x||95||20
+|35x (step 2-4)||95||20
 |-
 |||56||20
@@ Line 2,206: / Line 2,106: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Picking clones of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks</b></p>====
-<b> Kira</b> <br>
+<b>Investigator: Kira</b> <br>
 The plates were checked in the morning and they did not contain any visible colonies, thus the plates were kept @ 37C till the late afternoon in order to check again.
@@ Line 2,212: / Line 2,112: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>new ligation try and transformation of CD-biobrick</b></p>====
-<b> Kira</b> <br>
+<b>Investigator: Kira</b> <br>
 CD-biobrick plate contained just few (around 3 colonies), thus new ligation approach was performed.
@@ Line 2,218: / Line 2,118: @@
 Transformation was performed according to the standard protocol
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition of ViralBrick Ligations: 453 Z34C, 587 Z34C, 587 KO Z34C, 587 KO Z34C Spacer</b></p>====
@@ Line 2,292: / Line 2,191: @@
 Mini-Prep was performed according to the standard protocol
 <br />
-<li>P413 = pAAV_RC_CapIns_noRep clone1 c= 310,73 ng/µl
+<ul>
-<li>P414 = pAAV_RC_CapIns_noRep clone2 c= 217,66 ng/µl
+<li>P413 = pAAV_RC_CapIns_noRep clone1 c= 310,73 ng/µl</li>
-<li>P415 = pAAV_RC_CapIns_noRep clone3 c= 427,06 ng/µl
+<li>P414 = pAAV_RC_CapIns_noRep clone2 c= 217,66 ng/µl</li>
+<li>P415 = pAAV_RC_CapIns_noRep clone3 c= 427,06 ng/µl</li>
-<li>P416 = pSB1A2_EGFP_HQ clone1 c= 252,56 ng/µl
+<li>P416 = pSB1A2_EGFP_HQ clone1 c= 252,56 ng/µl</li>
-<li>P417 = pSB1A2_EGFP_HQ clone2 c= 255,02 ng/µl
+<li>P417 = pSB1A2_EGFP_HQ clone2 c= 255,02 ng/µl</li>
-<li>P418 = pSB1A2_EGFP_HQ clone3 c= 227,59 ng/µl
+<li>P418 = pSB1A2_EGFP_HQ clone3 c= 227,59 ng/µl</li>
+</ul>
 <br />
 {| border="1"
@@ Line 2,321: / Line 2,221: @@
 <br />
 <br />
-[[Image:Freiburg10 test digestion pAAV CapIns noRep pSB1A2 eGFP HQ.jpg|700px]]<br />
+[[Image:Freiburg10 test digestion pAAV CapIns noRep pSB1A2 eGFP HQ.jpg|thumb|center||700px]]<br />
 <br />
@@ Line 2,543: / Line 2,443: @@
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Competent XL1-B cells </b></p>====
-Investigator: Patrick
+'''Investigator: Patrick'''
 [[Image:Mistake.png|thumb|right|100px]]
@@ Line 2,603: / Line 2,503: @@
 |||95||120
 |-
-|8x (step 2-4||95||15
+|8x (step 2-4)||95||15
 |-
 |||60||10
@@ Line 2,689: / Line 2,589: @@
-[[Image:Freiburg10 Sequencing eGFP.jpg|600px]]
+[[Image:Freiburg10 Sequencing eGFP.jpg|thumb|center||650px]]
@@ Line 2,695: / Line 2,595: @@
 <p style="font-size:13px; color:red;"><b>Comment</b>: Since pAAV containing the synthetised Rep does not work out in cell culture an additional approach was performed inserting only the synthesized Cap and test this.</p>
-[[Image:Freiburg10 Sequencing pAAV RC CapIns noRep.jpg|600px]]
+[[Image:Freiburg10 Sequencing pAAV RC CapIns noRep.jpg|thumb|center||650px]]
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-prep and test digestion of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks</b></p>====
-Investigator: Kira<br/>
+'''Investigator: Kira<br/>'''
 Mini-Prep was performed with 3 clones of each construct in order to continue with test digestion and sequencing.<br/>
@@ Line 2,893: / Line 2,793: @@
 <br />
 Glycerol stocks were prepared:<br />
+<ul>
-<li>B371 = pAAV_RC_RepCapIns_SDMKpnI clone 1
+<li>B371 = pAAV_RC_RepCapIns_SDMKpnI clone 1</li>
-<li>B372 = pAAV_RC_RepCapIns_SDMKpnI clone 2
+<li>B372 = pAAV_RC_RepCapIns_SDMKpnI clone 2</li>
-<li>B373 = pAAV_RC_RepCapIns_SDMKpnI clone 3<br />
+<li>B373 = pAAV_RC_RepCapIns_SDMKpnI clone 3</li>
-<br />
+</ul><br />
 <br />
 Mini-Prep was performed according to the standard protocol<br />
 <br />
-<li>P431 = pAAV_RC_RepCapIns_SDMKpnI clone 1 = 362,21 ng/µl
+<ul>
-<li>P432 = pAAV_RC_RepCapIns_SDMKpnI clone 2 = 344,49 ng/µl
+<li>P431 = pAAV_RC_RepCapIns_SDMKpnI clone 1 = 362,21 ng/µl</li>
-<li>P433 = pAAV_RC_RepCapIns_SDMKpnI clone 3 = 331,30 ng/µl<br />
+<li>P432 = pAAV_RC_RepCapIns_SDMKpnI clone 2 = 344,49 ng/µl</li>
-<br />
+<li>P433 = pAAV_RC_RepCapIns_SDMKpnI clone 3 = 331,30 ng/µl</li>
+</ul><br />
 <br />
 Constructs were digested with KpnI for 70 minutes at 37°C, as controls the following constructs were used:<br />
-<li>pAAV_RC_1.2SDMSalI (P158)
+<ul>
-<li>pAAV_RC_CapIns_prepSDM (P396)
+<li>pAAV_RC_1.2SDMSalI (P158)</li>
-<li>pAAV_RC_CapIns_noRep (P413)
+<li>pAAV_RC_CapIns_prepSDM (P396)</li>
+<li>pAAV_RC_CapIns_noRep (P413)</li>
+</ul>
 {| border="1"
 | components   || align="right" |pAAV_RC_RepCapIns_SDMKpnI clone 1-3 || align="right" |pAAV_RC_1.2SDMSalI (P158) || align="right" |pAAV_RC_CapIns_prepSDM (P396) || align="right" |pAAV_RC_CapIns_noRep (P413)
@@ Line 2,928: / Line 2,830: @@
 <br />
 Preparation of gel:<br/>
-<br/>
+<ul><br/>
-<li>0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes<br/>
+<li>0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt, running time: 45 minutes</li><br/>
-<br />
+</ul><br />
-[[Image:Freiburg10 test digestion pAAV RC RepCapIns SDMKpnI.jpg|500px]]<br />
+[[Image:Freiburg10 test digestion pAAV RC RepCapIns SDMKpnI.jpg|thumb|center||600px]]<br />
 <p style="font-size:13px; color:red;"><b>Comment</b>: Test digestions look fine. Work will be continued with clone 2 (P432) which will be sent for sequencing tomorrow as well.</p>
@@ Line 3,046: / Line 2,948: @@
 <p style="font-size:13px; color:red;"><b>Comment</b>: A site-directed mutagenesis has to be performed in order to obtain the plasmids which can be co-transfected with the modified construct containing the targeting molecules. Aim is to knockout VP1 for VP1-insertion approaches and VP2 (knockout) for VP-2 fusion. This site-directed mutagenesis has to be performed at several constructs:</p><br />
-<b>Knock-out of VP1:</b><br />
+<b>Knock-out of VP1:</b>
 <br />
-<li>pAAV_RC_1.2SDMSalI (P158)
+<ul>
-<li>pAAV_RC_CapIns_prepSDM (P396)
+<li>pAAV_RC_1.2SDMSalI (P158)</li>
-<li>pAAV_RC_CapIns_noRep (P413)
+<li>pAAV_RC_CapIns_prepSDM (P396)</li>
-<li>pAAV_RC_RepCapIns_SDMKpnI (P432)
+<li>pAAV_RC_CapIns_noRep (P413)</li>
+<li>pAAV_RC_RepCapIns_SDMKpnI (P432)</li>
+</ul>
 <br /><br />
-<b>Knock-out of VP2:</b><br />
+<b>Knock-out of VP2:</b>
 <br />
-<li>pAAV_RC_1.2SDMSalI (P158)
+<ul>
-<li>pAAV_RC_CapIns_prepSDM (P396)
+<li>pAAV_RC_1.2SDMSalI (P158)</li>
-<li>pAAV_RC_CapIns_noRep (P413)
+<li>pAAV_RC_CapIns_prepSDM (P396)</li>
-<li>pAAV_RC_RepCapIns_SDMKpnI (P432)<br />
+<li>pAAV_RC_CapIns_noRep (P413)</li>
+<li>pAAV_RC_RepCapIns_SDMKpnI (P432)</li>
+</ul>
 <br />
 <b>Used primers:</b><br />
 <b>Knock-out of VP1:</b><br />
-<li>primer forward: VP1-M1L_for (O170)
+<ul>
-<li>primer reverse: VP1-M1L_rev (O171)<br />
+<li>primer forward: VP1-M1L_for (O170)</li>
+<li>primer reverse: VP1-M1L_rev (O171)</li>
+</ul>
 <b>Knock-out of VP2:</b><br />
-<li>primer forward: VP2-ko_for (O165)
+<ul>
-<li>primer reverse: VP2-ko_rev (O166)<br />
+<li>primer forward: VP2-ko_for (O165)</li>
+<li>primer reverse: VP2-ko_rev (O166)</li>
+</ul>
 <br />
 <b>Used plasmid and water per approach:</b>
@@ Line 3,132: / Line 3,042: @@
 Transformation was performed according to standard protocol using XL10-Gold cells. Cells were plated on plate containing Ampicilin.
 <p style="font-size:13px; color:red;"><b>Comment</b>: There were not enough cells for all approaches. Therefore, only transformation of approach 5 and 8 were performed using 45 µl of cells. The other approaches were conducted with 30 µl.</p><br />
 ===<p style="font-size:17px; background-color:#00dd77;">119. labday 13.09.2010</p>===
@@ Line 3,177: / Line 3,086: @@
 .5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 90 Volt, running time: ~90 minutes
 <br>
-[[Image:Freiburg10 pCerulean VP1up NLS targeting VP2 3.jpg|600px]]<br />
+[[Image:Freiburg10 pCerulean VP1up NLS targeting VP2 3.jpg|thumb|center||600px]]<br />
 <br />
 <b>Gel-Extraction:</b><br />
@@ Line 3,423: / Line 3,332: @@
 | 10 mM dNTP mix||  align="right" |1 ||  align="right" |1
 |-
-| forward primer: O93 ||  align="right" |2,5 ||  align="right" |2,5
+| forward primer: O89 ||  align="right" |2,5 ||  align="right" |2,5
 |-
 | reverse primer: O120 ||  align="right" |2,5 ||  align="right" |2,5
@@ Line 4,130: / Line 4,039: @@
 <ul>
-<li><b>P432 = pAAV_RC_RepCapIns_SDMKpnI</b> = <b>AM_3</b></li>
+<li>P432 = pAAV_RC_RepCapIns_SDMKpnI = AM_3</li>
 <li>Used primers: 1250 for</li>
 </ul>
@@ Line 4,136: / Line 4,045: @@
 <b> Results:</b>
-[[Image:Freiburg10 Sequencing pAAV RC RepCapIns SDMKpnI.jpg|700px]]
+[[Image:Freiburg10 Sequencing pAAV RC RepCapIns SDMKpnI.jpg|thumb|center||750px]]
 ====<p style="font-size:15px; background-color:#66bbff;"><b> Repetition of the fusion of VP2/3 to C-terminus of Zegfr:1907_"Linker", CFP_MiddleLinker and 6xHis_MiddleLinker construct </b></p>====
 <b>Investigator: Anissa</b>
@@ Line 4,562: / Line 4,472: @@
 Preparation of gel:<br/>
 <br/>
-<li>0,4 g Agarose, 50 ml TAE (0,8%), 3 µl GELRED , at 110 Volt, running time: 45 minutes<br/>
+<ul>
+<li>0,4 g Agarose, 50 ml TAE (0,8%), 3 µl GELRED , at 110 Volt, running time: 45 minutes</li>
+</ul>
 <br />
-[[Image:Freiburg10 test digestion pCerulean VP1up NLS targeting VP2 3.jpg|700px]]<br />
+[[Image:Freiburg10 test digestion pCerulean VP1up NLS targeting VP2 3.jpg|thumb|center||700px]]<br />
 <p style="font-size:13px; color:red;"><b>Comment</b>: it is still not clear whether or not the correct insert was cloned into pCerulean. Therefore, P475 and P473 will be sent for sequencing tomorrow using pEGFP_C2-RP as primer to verify VP2/3 insertion.</p>
@@ Line 4,582: / Line 4,494: @@
 <li>P463</li>
 </ul>
 ===<p style="font-size:17px; background-color:#00dd77;">121. labday 15.09.2010</p>===
@@ Line 4,599: / Line 4,512: @@
 *Hybridisatzion of oligos
-**453 Z34C: 143 & 144
+**11:453 Z34C: 143 & 144
-**587 Z34C: 144 & 145
+**12:587 Z34C: 145 & 146
-**587 KO Z34C:
+**13:587 KO Z34C: 147 & 148
+**14:587 KO Z34C: 149 & 150
+Condtitions as before:
+*2 µg DNA, (3,3 µl of each oligo)
+*2 µl Klenow buffer
+*0,1 µl dNTPs (1µl of 1:10 dilution of our aliquots)
+*9,8 µl H2O
+Total volume: 20 µl
+Hybridisation was carried out on the gradient cycler using the program "tkfill"
+,5 µl Klenow Fragment was added after the temperature reached 37°C. Klenow reaction was carried out for 1 h in the 37°C room.
+Afterwards, the samples were purified with the Quiagen PCR Purification kit. Elution in 50 µl H2O.
+Nanodrop concentrations:
+*11: 47.08 ng/µl
+*12: 28.50 ng/µl
+*13: 37.69 ng/µl
+*14: 42.98 ng/µl
+µl of SspI & SalI for sample 11/ BamHI & PvuII for samples 12,13,14 as well as 5,8 µl buffer 4 for each sample were added to the purified samples. Digestion for 3 h at 37 °C
+The vector backbone pSB1C3_VCK_BLA (P320)was also digested with BamHI/PvuII and SspI/SalI for 3 h. Afterwards,the cut vector fragment was dephosphorylated with Antarctic Phosphatase for 1h at 37°C.
+The oligos and the vector fragments were mixed with loading dye and run over a preparative gel (1%):
+[[Image:Freiburg10 15092010 vb.png|thumb|400px|none|Oligos ran between 100 - 200 bp, the unhybridized oligo control ran a bit further.]]
+Fragments were cut out, followed by gel extraction. Two ligations were prepared: T4-Ligation and Quickligation.
+Quickligation: 10 min, followed by trafo. Plates were incubated overnight.
+T4-Ligation: Overnight
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning VP2/3 approaches into pSB1C3_VCK vector</b></p>====
@@ Line 4,672: / Line 4,619: @@
 <b>Investigator: Kira </b>
+<p style="color:#66bbff;">Comments: Since the last approach with the CD did not work, we performed a colony PCR in order to detect the positive clones which should contain the CD gene. We used the two SDM primers, originally used for deleting the iGEM restriction sites in the CD gene, which should only bind to the CD sequence.</p>
+<br />
 colonies were picked from the plate and dispersed first into the PCR tube and then into DYT media as a backup in case of successful PCR reaction.
 {| border="1"
@@ Line 4,719: / Line 4,668: @@
 [[Image:Freiburg10 colony pcr 2010_09_15.jpg|250px]]<br />
+<br />
+According to the gel, clone 3, 6, 7, 9, 11, 14 contain CD gene. These samples will be incubated over-night @ 37C.
-According to the gel, clone 3, 6, 7, 9, 11, 14 contain CD gene. These samples will be incubated over-night @ 37C
+<br />
+<p style="color:#ff3300;"><b>Comment added on 17.09.2010 :</b> We (Bea, Kira, Stefan and Tobi) wondered why sequencing of one of the positive clones of the colony PCR revealed that the construct did not contain the expected CD gene but CFP instead. Today, we realized that the colony PCR does not reveal <b>only</b> positive constructs which contain the cytosine deaminase. Problem is that we received the CD gene originating from the COD-BA gene of <i>E. coli</i>. Since we performed a colony PCR with XL1-B <i>E. coli </i> cells the used primers will bind to the genomic CD-gene aswell which could explain the positive clones in the gel, the negative results of seuqencing and another test digest of hte sequenced clone.
+<br />
+<b>Next steps</b> are that we are performing the colony PCR again with new primers which means that we are using the iGEM primers which should specifically bind to the constructs which contain the iGEM prefix and suffix add-ons. Since CFP does contain the iGEM restriction sites aswell, but differ in the size of the expected fragment (CD ~1100bp vs. CFP ~700bp) the positive clones should be detectable by the size length.</p>
 <br />
@@ Line 4,757: / Line 4,711: @@
 ,5 g Agarose,50 ml TAE (1%), 5 µl Gelred , at  Volt
 <br />
+[[Image:Freiburg10 gelex affibody vp23.jpg|250px]]<br />
 '''Gelextraction:'''<br />
@@ Line 5,032: / Line 4,986: @@
 ,4 g Agarose,50 ml TAE (0,8%), 3 µl Gelred , at 110 Volt; run for ~40 minutes
 <br />
-[[Image:Freiburg10 Biobrick PCR of RepCap.jpg|500px]]<br />
+[[Image:Freiburg10 Biobrick PCR of RepCap.jpg|thumb|center||550px]]<br />
 For vector:<br />
 ,5 g Agarose,50 ml TAE (1%), 3 µl Gelred , at 110 Volt; run for ~50 minutes<br />
-[[Image:Freiburg10 Biobrick vector digest of RepCap.jpg|500px]]<br />
+[[Image:Freiburg10 Biobrick vector digest of RepCap.jpg|thumb|center||550px]]<br />
 <br />
 <br />
@@ Line 5,169: / Line 5,123: @@
 <li>pAAV_RC_RepCapIns_SDMKpnI_VP2-ko clone 1 (P463)</li>
 </ul>
 <p style="font-size:13px; color:red;"><b>Comment</b>:All sequences showed that the site directed mutagenesis worked out for all constructs. All sequencing results revealed problems in the area shown below:</p>
-[[Image:Freiburg10 Sequencing VP1 2 ko problem(2).jpg|500px]]<br />
+[[Image:Freiburg10 Sequencing VP1 2 ko problem(2).jpg|thumb|center||500px]]<br />
 <br />
-[[Image:Freiburg10 Sequencing VP1 2 ko problem(1).jpg|500px]]<br />
+[[Image:Freiburg10 Sequencing VP1 2 ko problem(1).jpg|thumb|center||500px]]<br />
 ===<p style="font-size:17px; background-color:#00dd77;">122. labday 16.09.2010</p>===
+====<p style="font-size:15px; background-color:#66bbff;"><b>Trafo of T4 ligation, Results of Quickligation</b></p>====
+<b>Investigator: Achim</b>
+<br />
+No clones on the Quickligation plates. Trafo of T4 Ligation was carried out in XL1B cells. Results will be evaluated tomorrow.
+====<p style="font-size:15px; background-color:#66bbff;"><b>Mini Prep and test digestion of several constructs</b></p>====
+<b>Investigator: Anissa, Bea</b>
+<p style="color:#66bbff;"><i>Comment:</i> Several test digestions have been performed for verifying the cloned fragment into the plasmid backbone. The constructs for reassembling the vector plasmid (pSB1C3_leftITR_PROMOTER_betaglobin_GOI) were digested with EcoRI and NgomIV, the SV40 construct was digested with EcoRI and PstI. </p>
+<br />
+<b>Test digestion of the constructs:</b>
+<ul>
+<li>pSB1C3_leftITR_phTERT_betaglobin_mVenus (P485-486)</li>
+<li>pSB1C3_leftITR_phTERT_betaglobin_mGMK (P483-484) </li>
+<li>pSB1C3_leftITR_pCMV_betaglobin_mGMK (P487-488)</li>
+<li>pSB1C3_SV40 (P493-494)</li>
+</ul>
+<br />
+<b>Expected size fragments are: </b>
+<ul>
+<li>(P485-486): 1150bp, 2780bp</li>
+<li>(P483-484): 1150bp, 2650bp </li>
+<li>(P487-488): 1300bp, 2600bp </li>
+<li>(P493-494): 270bp, 2030bp</li>
+</ul>
+<br />
+<b>Results:</b> Test digestion of the constructs looks well, except for the construct 494 which is one clone of the construct pSB1C3_SV40. Since the other clone is fine, the construct was discarded. The control K437 is the plasmid pSB1C3_leftITR_hTERT_beta-globin without the assembled GOI (mGMK and mVenus repsectively). The GOI´s are in the RFC25 standard therefore leading to scar with a additional NgoMIV after fusing it to the before mentioned construct which is in the RFC10 standard. Therefore, test digestion was carried out with NgoMIV and EcoRI leading to fragments which can be separated from the backbone in a 1% agarose gel. The control vector should only be linearized because it does not contain any NgomIV rstriction site.
+<br />
+[[Image:Freiburg10 pSB1C3 leftITR Promoter betaglob GOI 16 09 2010.jpg|thumb|center|500px]]
 ====<p style="font-size:15px; background-color:#66bbff;"><b>Test digestion of pSB1C3_VP123</b></p>====
 <b>Investigator: Jessica<br /></b>
@@ Line 5,221: / Line 5,205: @@
 <br/>
-[[Image:Freiburg10_Test digestion_VP123.jpg|200px]]
+[[Image:Freiburg10_Test digestion_VP123.jpg|500px]]
 <br />
 *<b>Expected fragment sizes:</b> <br/>
 bp<br/>
 bp<br/>
-<p style="font-size:13px; color:#003399;"><b>Comment</b>: Samples were sent for sequencing.  </p>
+<p style="font-size:13px; color:#003399;"><b>Comment</b>: Sample P489 was sent for sequencing with primer VR2.
+Sequencing result looks good, VP123 is in </p>
+====<p style="font-size:15px; background-color:#66bbFF;"><b>Continuation of preparation of competent E.coli</b></p>====
+'''Investigator: Jessica'''<br>
+preparation of competent BL21 was finished according to the standard protocol
+<ul><li>aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer</li></ul>
+don't use before results of testtrafo is writen down <br>
+testtrafo was made with pUC and Amp
+====<p style="font-size:15px; background-color:#66bbff;"><b>Mini prep of CD-clones, which were identified by colony pcr</b></p>====
+<b>Investigator: Kira<br /></b>
+c(clone1) = 160,29 ng/ul <br />
+c(clone2) = 167,98 ng/ul <br />
+c(cloone3) = 181, 51 ng/ul <br />
+clone 3 was sent for sequencing <br />
+====<p style="font-size:15px; background-color:#66bbff;"><b>Repetition: PCR for biobrick production of P40 promotor</b></p>====
+<b>Investigator: Stefan<br /></b>
+<p style="font-size:13px; color:red;"><b>Comment</b>: During trouble-shooting we realized that the reverse primer P40_rev (O153) was designed forward and therefore does not work. A new primer will have to be designed and ordered to repeat the PCR.</p>
+<br/>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Results of Colony PCR</b></p>====
+<b>Investigator: Hanna<br /></b>
+<br/>
+<b>Comment:</b> In order to find out, whether the VP2/3 or the VP2/3_Capins fragment was successfully cloned into pCerulean_Zegfr:1907_"Linker" (--> Linker library), pCerulean_6xHis_MiddleLinker and pCerulean_CFP_MiddleLinker, a colony PCR was performed yesterday. From each plate 2-4 clones were picked, so that we got 41 samples (including one positive control = pAAV_RC). With VP_3500_for and VP_4200_rev primer a 880 fragment should be amplified of each colony, if cloning functioned. <br/>
+<br/>
+Samples were loaded onto a 1% agarose gel: <br/>
+<br/>
+* 1. pCerulean_Zegfr:1907_ShortLinker_VP2/3
+* 2. pCerulean_Zegfr:1907_MiddleLinker_VP2/3_Capins
+* 3. pCerulean_Zegfr:1907_LongLinker_VP2/3
+* 4. pCerulean_6xHis_MiddleLinker_VP2/3
+* 5. pCerulean_Zegfr:1907_SEG_VP2/3
+* 6. pCerulean_6xHis_MiddleLinker_VP2/3_Capins
+* 7. pCerulean_CFP_MiddleLinker_VP2/3_Capins
+* 8. pCerulean_Zegfr:1907_ShortLinker_VP2/3_Capins
+* 9. pCerulean_ Zegfr:1907_SEG_VP2/3_Capins
+* 10. pCerulean_Zegfr:1907_LongLinker_VP2/3_Capins
+* 11. pCerulean_Zegfr:1907_MiddleLinker_VP2/3
+<br/>
+[[Image:Freiburg10 ColonyPCR15 09 10.png]] <br/>
+<br/>
+As can be seen in the gel picture, it seemed that just clone 9.3, 10.2, 11.1 and 11.2 were VP2/3 positive. Curiously the positive control (K) was negative. <br/>
+<br/>
+These 4 clones were mini-preped by Achim and sent for sequencing to GATC: primer = GATC_std_CMV-F. <br/>
+<br/>
+Because I found out that some things went wrong during VP cloning due to false geneious annotations and theoretical cloning approaches, all pAAV_RC constructs (with/without Cap or Rep-insert, 4-fold mutated, PvuII insertion, KpnI-backmutation,...) were theoretically prepared again (see geneious folder: "pAAV_RC constructs (DO NOT CHANGE!!!)"). <br/>
+In addition to that the theoretical cloning of the final N-terminal fusion approaches to VP2/3 were performed once again with these correct sequences. With the help of Sven cloning of the not yet positive approaches were repeated (The ones which were negative in colony PCR). <br/>
+<br/>
+<b>To do (tomorrow):</b> Pciking clones, analyze sequencing. Check and plan HSPG-KO approaches and Zegfr:1907
+<br/>
+<p style="font-size:15px; font-weight: bold; color: blue;">Practical Cloning:</p>
+**Vector: pCerulean_CFP_MiddleLinker (P407), pCerulean_Zegfr:1907_MiddleLinker (P408), pCerulean_Zegfr:1907_SEG (P409), pCerulean_Zegfr:1907_ShortLinker (P410), pCerulean_Zegfr:1907_LongLinker (P412), pCerulean_6xHis_MiddleLinker (P374)
+**Insert: pSB1C3_VP2/3 (P440), pSB1C3_VP2/3_ins_cap (P444)
+**new vector name: pCerulean_CFP_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_MiddleLinker_VP2/3, pCerulean_Zegfr:1907_SEG_VP2/3, pCerulean_Zegfr:1907_ShortLinker_VP2/3, pCerulean_Zegfr:1907_LongLinker_VP2/3, pCerulean_6xHis_MiddleLinker_VP2/3 AND pCerulean_CFP_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_MiddleLinker_VP2/3-insCap, pCerulean_Zegfr:1907_SEG_VP2/3-insCap, pCerulean_Zegfr:1907_ShortLinker_VP2/3-insCap, pCerulean_Zegfr:1907_LongLinker_VP2/3-insCap, pCerulean_6xHis_MiddleLinker_VP2/3-insCap
+*buffer used:4 <br/>
+<b>Comment</b> Because the VP2/3 frament has approximately the same size as the backbone pSB1C3 it may be difficult to separate them on the agarose gel. Therefore a single cutter enzyme (MscI) was chosen to digests the backbone. This enzyme also works in buffer 4 + BSA and needs to be incubated at 37°C.
+**Vector: AgeI, SpeI
+**Insert: NgoMIV, SpeI, SacI
+<br/>
+<br />
+<p style="font-size:15px; font-weight: bold; color: blue;">Digestion</p>
+<br/>
+[[Image:Freiburg10 Digestion16 09003.jpg|500px]]
+*Incubation: 2 h
+<br/>
+<p style="font-size:15px; font-weight: bold; color: blue;">Agarose gel</p>
+<br/>
+[[Image:Freiburg10 N-terminalFKlappe2.png|500px|thumb|center|Green boxes were cut out.]]
+<br/>
+Gelex, ligation and trafo were performed according to standard protocol. <br/>
+<b>Next step:</b> Colony PCR of each ligation approach.
+===<p style="font-size:17px; background-color:#00dd77;">122. labday 17.09.2010</p>===
+====<p style="font-size:15px; background-color:#66bbff;"><b>Colony PCR of N-terminal fusion approach </b></p>====
+<b>Investigator: Achim</b>
+<br/>
+<p style="color:#66bbff;">Comment: </p>
+[[Image:Freiburg10 17 9 colonyPCR.png|thumb|400px|none|]]
+====<p style="font-size:15px; background-color:#66bbff;"><b>Sequencing analysis of pSB1C3_VP123</b></p>====
+<b>Investigator: Bea</b>
+<br/>
+<p style="color:#66bbff;">Comment: The constrcuct was created by performing a PCR with the pAAV_RC_inscap construct. The sequence analysis reveals that the suffix looks well. Additionally the mutation from TAA to TGA can be seen.</p>
+[[Image: Freiburg10_Seqanalysis_pSB1C3_VP123_inscap.jpg|thumb|center|700px]]
+====<p style="font-size:15px; background-color:#66bbff;"><b>ViralBricks: Picking clones from T4 ligation</b></p>====
+<b>Investigator: Achim</b>
+<br/>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Sequencing results N-terminal fusion - part 1</b></p>====
+<b>Investigator: Hanna</b>
+<br/>
+<b>Comment:</b> Colony PCR delivered that 4 clones contained the VP2/3 or VP2/3_Capins sequence. These clones were sent for sequencing:
+<br/>
+<br/>
+<b>1. pCerulean_Zegfr:1907_LongLinker__VP2/3_Capins:</b> <br/>
+[[Image:Freiburg10 pCer LL Aff.png|600px|thumb|center|]] <br/>
+<br/>
+Sequencing looked well: VP2/3_Capins was successfully fused to the C-terminus of the Long-Linker. The additional basepair at the beginning of the Affibody was misinterpreted (as one can see in the chromatogram) by Geneious. <br/>
+<br/>
+<b>2. pCerulean_Zegfr:1907_MiddleLinker__VP2/3 clone 1:</b> <br/>
+[[Image:Freiburg10 pCer ML Aff.png|600px|thumb|center|]] <br/>
+<br/>
+Sequencing looked well: VP2/3 was successfully fused to the C-terminus of the Middle-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious. <br/>
+<br/>
+<b>3. pCerulean_Zegfr:1907_MiddleLinker__VP2/3 clone 2:</b> <br/>
+[[Image:Freiburg10 pCer ML Aff 2.png|600px|thumb|center|]] <br/>
+<br/>
+Sequencing looked well: VP2/3 was successfully fused to the C-terminus of the Middle-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious. <br/>
+<br/>
+<b>4. pCerulean_Zegfr:1907_SEG__VP2/3_Capins:</b> <br/>
+[[Image:Freiburg10 pCer SEG Aff.png|600px|thumb|center|]] <br/>
+<br/>
+Sequencing looked well: VP2/3_Capins was successfully fused to the C-terminus of the SEG-Linker. The additional basepair at the beginning of the Affibody was again misinterpreted (as one can see in the chromatogram) by Geneious. <br/>
+<br/>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep and test digestion of several constructs</b></p>====
+<b>Investigator: Stefan</b>
+<b>Glycerol stocks were prepared:</b><br />
+<ul>
+<li>B430 =	pSB1C3_001_RC_4fold mutated clone 1</li>
+<li>B431 =	pSB1C3_001_RC_4fold mutated clone 2</li>
+<li>B432 =	psB1C3_001_RC_InsRepCap clone 1</li>
+<li>B433 =	psB1C3_001_RC_InsRepCap clone 2</li>
+<li>B434 =	pSB1C3_001_RC_InsCap clone 1</li>
+<li>B435 =	pSB1C3_001_RC_InsCap clone 2</li>
+<li>B436 =	pSB1C3_001_RC_InsRepCap_KpnIback clone 1</li>
+<li>B437 =	pSB1C3_001_RC_InsRepCap_KpnIback clone 2</li>
+<li>B438 =	pSB1C3_001_VP2/3insCap clone 1</li>
+<li>B439 =	pSB1C3_001_VP2/3insCap clone 2</li>
+<li>B440 =	pSB1C3_ZEGFR:1907_VP2/3 clone 1</li>
+<li>B441 =	pSB1C3_ZEGFR:1907_VP2/3 clone 2</li>
+<li>B442 =	pSB1C3_GFP_RFC25_upper band clone 1</li>
+<li>B443 =	pSB1C3_GFP_RFC25_upper band clone 2</li>
+<li>B444 =	pSB1C3_GFP_RFC25_lower band clone 1</li>
+<li>B445 =	pSB1C3_GFP_RFC25_lower band clone 2</li>
+<li>B446 =	pSB1C3_GFP_RFC25 clone 1</li>
+<li>B447 =	pSB1C3_GFP_RFC25 clone 2</li>
+</ul>
+<br />
+<br />
+<b>Mini-Prep was performed according to the standard protocol</b><br />
+<br />
+<ul>
+<li>P506	pSB1C3_001_RC_4fold mutated clone 1</li>
+<li>P507	pSB1C3_001_RC_4fold mutated clone 2</li>
+<li>P508	psB1C3_001_RC_InsRepCap clone 1</li>
+<li>P509	psB1C3_001_RC_InsRepCap clone 2</li>
+<li>P510	pSB1C3_001_RC_InsCap clone 1</li>
+<li>P511	pSB1C3_001_RC_InsCap clone 2</li>
+<li>P512	pSB1C3_001_RC_InsRepCap_KpnIback clone 1</li>
+<li>P513	pSB1C3_001_RC_InsRepCap_KpnIback clone 2</li>
+<li>P514	pSB1C3_001_VP2/3insCap clone 1</li>
+<li>P515	pSB1C3_001_VP2/3insCap clone 2</li>
+<li>P516	pSB1C3_ZEGFR:1907_VP2/3 clone 1</li>
+<li>P517	pSB1C3_ZEGFR:1907_VP2/3 clone 2</li>
+<li>P518	pSB1C3_GFP_RFC25_upper band clone 1</li>
+<li>P519	pSB1C3_GFP_RFC25_upper band clone 2</li>
+<li>P520	pSB1C3_GFP_RFC25_lower band clone 1</li>
+<li>P521	pSB1C3_GFP_RFC25_lower band clone 2</li>
+<li>P522	pSB1C3_GFP_RFC25 clone 1</li>
+<li>P523	pSB1C3_GFP_RFC25 clone 2</li>
+<li>P524	pSB1C3_VP2/3</li>
+<li>P525	pSB1C3_VP2/3_ins_cap</li>
+</ul><br />
+<br />
+<br />
+<b>Test digestion:</b><br />
+Test digestion was performed for plasmids P506, P507, P510 to P513 and for P522 and P523. Test digestion of P514 to P521 will be performed tomorrow. Preps for P524 and P525 were inocculated from glycerol stock and therefor no digestion will be performed.
+The constructs were digested using XbaI and AgeI for 40 minutes at 37°C.<br />
+{| border="1"
+| components   || align="right" |P470-P481|| align="right" |P470-P481
+|-
+| DNA  ||  align="right" |2 ||  align="right" |2
+|-
+| Buffer 4 (10x)||  align="right" |1 ||  align="right" |1
+|-
+|XbaI ||  align="right" |0,4 ||  align="right" |0,4
+|-
+|AgeI ||  align="right" |0,4||  align="right" |0,4
+|-
+|H<sub>2</sub>O||  align="right" |7,2||  align="right" |6,2
+|-
+|'''Total volume '''||  align="right" |10 ||  align="right" |10
+|}
+<br />
+Preparation of gel:<br/>
+<br/>
+<ul>
+<li>0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 110 Volt, running time: 40 minutes</li>
+</ul>
+<br />
+[[Image:Freiburg10 test digestion pSB1C3 001 RepCaps pSB1C3 GFP RFC25.jpg|thumb|center|700px]]<br />
+<p style="font-size:13px; color:red;"><b>Comment</b>: </p>
+<b>Sent for sequencing:</b><br />
+Primer used:<br />
+VR2<br />
+plasmid no:<br />
+P522<br />
+tube labeled: SB_1
+====<p style="font-size:15px; background-color:#66bbff;"><b>Sent for sequencing</b></p>====
+<b>Investigator: Stefan</b><br />
+pSB1C3_SV40 (P493) was sent for sequencing using VR2 primer. Results have to be checked tomorrow.
+====<p style="font-size:15px; background-color:#66bbff;"><b>Sequencing analysis of VP2/3 insertion into pCerulean_VP1up_NLS_targeting motif</b></p>====
+<b>Investigator: Stefan</b><br />
+<b>Comment:</b> Sequences can be found in GATC Watchbox (date 15.10.10) labeled SB_1 (VP2/3 insertion) and SB_2 (VP2/3_insCap insertion).
+[[Image:Freiburg10 Sequencing VP2 3 insertion.JPG|thumb|center|750px]]<br />
+<br />
+[[Image:Freiburg10 Sequencing VP2 3 insCap insertion.JPG|thumb|center|750px]]
+<br/>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Biobrick production of VP2/3- and VP2/3 cap-Gsat_linker </b></p>====
+Motivation: Production of fusion proteins
+<b>Investigator: Kira</b><br>
+<b>Digestions of backbones and inserts:</b>
+{| border="1"
+| align="left" | '''Components''' ||align="left"| VP2/3 ||align="left"| VP2/3 cap ||align="left"| Gsat linker
+|-
+| align="left" | DNA  ||align="left"| 7 ||align="left"| 7 ||align="left"| 7
+|-
+| align="left" | BSA (100x) ||align="left"| 0||align="left"| 0 ||align="left"|0
+|-
+| align="left" | Buffer no. 4 (10x) ||align="left"| 2,5 ||align="left"|2,5 ||align="left"| 2,5
+|-
+| align="left" | EcoRI ||align="left"| 0,5||align="left"| 0,5 ||align="left"| 0,5
+|-
+| align="left" | NgoMIV ||align="left"| 0,5 ||align="left"| 0,5 ||align="left"|0
+|-
+| align="left" |XbaI  ||align="left"| 0 ||align="left"|0 ||align="left"|1,25
+|-
+| align="left" | H<sub>2</sub>O ||align="left"| 14,5 ||align="left"|14,5 ||align="left"|13,75
+|-
+| align="left" | '''Total volume''' ||align="left"| 25||align="left"| 25 ||align="left"|25
+|}
+<br />
+incubation @ 37 C for approx. 3 h
+% agarose gel <br />
+[[Image:Freiburg10_2010_09_18.jpg|thumb|center|500px]]
+<b>Ligation:</b> <br />
+T4 ligase was used. ligation over night @ 18 C <br />
+VP2/3-mix 8 ul (3,7 ul vector + 4,3 ul insert) <br />
+VP2/3 cap-mix 8 ul (3,7 ul vector + 4,3 ul insert) <br />
+===<p style="font-size:17px; background-color:#00dd77;">123. labday 18.09.2010</p>===
+====<p style="font-size:15px; background-color:#66bbFF;"><b>Continuation of preparation of competent E.coli</b></p>====
+'''Investigator: Jessica'''<br>
+preparation of competent XL1blue was finished according to the standard protocol
+<ul><li>aliquots of 60µl (to use for 1 trafo) are stored in -80°C freezer</li></ul>
+don't use before results of testtrafo is writen down <br>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Test digestion of pSB1C3_ZEGFR:1907_VP2/3</b></p>====
+<b>Investigator: Jessica<br /></b>
+<br>
+{| border="1"
+| '''Samples || Plasmid  || Clone || Concentration [ng/µl]
+|-
+| '''P514  || pSB1C3_001_VP2/3insCap    ||1 ||318.9
+|-
+| '''P515  || pSB1C3_001_VP2/3insCap    ||2 ||321.8
+|-
+| '''P516  || pSB1C3_ZEGFR:1907_VP2/3   ||1 ||191,6
+|-
+| '''P517 ||  pSB1C3_ZEGFR:1907_VP2/3  ||2||210,8
+|-
+| '''P518 ||  pSB1C3_GFP_RFC25_upper band  ||1||138.3
+|-
+| '''P519 || pSB1C3_GFP_RFC25_upper band  ||2||72.3
+|-
+| '''P520 || pSB1C3_GFP_RFC25_lower band ||1||141.6
+|-
+| '''P521 ||  pSB1C3_GFP_RFC25_lower band  ||2||127.5
+|}
+<br />
+<b>Test digestion:</b>
+<br>
+{| border="1"
+| components ||Volume for each sample P516/517 /µl ||Volume for each sample P514/515 /µl||Volume for each sample P518-521 /µl
+|-
+| DNA  || 2,5|| 3,5|| 1,5
+|-
+| BSA (10x) || 1|| 1|| 1
+|-
+| Buffer 1/4/4 (10x) || 1 || 1 || 1
+|-
+|Enzyme NgoMIV ||0,5 ||- ||-
+|-
+|Enzyme DraIII ||0,5 ||- ||-
+|-
+|Enzyme XbaI ||- ||- ||0,4
+|-
+|Enzyme AgeI ||- ||- ||0,4
+|-
+|Enzyme MscI ||- ||0,4 ||-
+|-
+|Enzyme SpeI ||- ||0,4 ||-
+|-
+|H2O || 4,5|| 3,7|| 5,7
+|-
+|'''Total volume /µl'''|| 10|| 10|| 10
+|}
+<br />
+<b>Preparation of gel:</b><br/>
+<br/>
+*Incubation time: 1,5 h, Incubation temperature: 37°<br/>
+*0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt
+<br/>
+[[Image:Freiburg10_Test digestion_P514-521.jpg|500px]]
+<br />
+*<b>Expected fragment sizes:</b> <br/>
+P516/517: 3540bp / 651bp<br/>
+P514/515: 1300bp / 2700bp<br>
+P518-521: 2000bp / 700bp <br>
+<p style="font-size:13px; color:#003399;"><b>Comment</b>: All samples look OK! Samples will be sent for sequencing on monday.  </p>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Mini-Prep of pSB1C3_SV40 clone 1 (B420)</b></p>====
+<b>Investigator: Jessica<br /></b>
+was done because of the low concentration of the plasmid P493
+*P526: pSB1C3_SV40 clone 1 (=B420)  c= 176,0 ng/µl<br>
+====<p style="font-size:15px; background-color:#66bbFF;"><b>New aliquots (60µl) of BL21 can be used !  (Jessica)</b></p>====
+<br/>
+<br/>
+====<p style="font-size:15px; background-color:#66bbFF;"><b>New Colony PCR of N-terminal fusion approach</b></p>====
+<b>Investigator: Hanna<br /></b> <br/>
+<br/>
+<b>Comment:</b> Because just 3 clones of the colony PCR (yesterday) were VP2/3 positive, new colonies were picked for a colony PCR. We assumed that we can find some positive clones, because the 3 successful ligations contained VP2/3 AND VP2/3_Capins, so that we concluded, that digestion worked and that we just have to search for positive clones on the plates. <br/>
+<br/>
+<b>A mastermix for 28 samples was prepared: </b><br/>
+* 10x Taq Buffer: 28 µL
+* MgCl<sub>2</sub> (50 mM): 2.8 µL
+* dNTPs: 5.6 µL
+* Primer 1 (4200 rev = O42): 5.6 µL
+* Primer 2 (3500 for = O39): 5.6 µL
+* Taq polymerase: 1.4 µL
+* H<sub>2</sub>O: 231 µL
+--> Volume for each reaction: 10 µL
+<br/>
+<br/>
+colonies were picked with a tooth-pick from each plate (6), inoculated into the 10 µL mastermix and then put into 5 µL DYT containing Kanamycin. <br/>
+As positive control 1 ng of P158 = pAAV_RC_SDM_SalI and as negative control 1 ng of P42 = pAAV_RFC25 were used. <br/>
+<br/>
+<b>PCR program:</b> <br/>
+* 95°C, 6'
+x
+* 95°C, 25''
+* 50.5°C, 30''
+* 68°C, 54''
+x
+* 68°C, 5'
+Hold 4°C
+<br/>
+<br/>
+<b>Gel picture:</b> <br/>
+[[Image:Freiburg1 CPCR allpos002.jpg|thumb|center|500px]]
+<br/>
+All PCR reactions delivered positive VP2/3 results (expected size: 880 bp). Unfortunately also the negative control was positive. We decided that 2 clones of each plate will be mini-preped. The remaining 2 clones will be pelletized and stored at -20°C. On monday several test digestions will be performed (perhaps 1 of each plate).
+<br/>
+====<p style="font-size:15px; background-color:#66bbFF;"><b>Trafo of pSB1C3_GSAT_VP2/3 and pSB1C3_GSAT_VP2/3_Capins</b></p>====
+<b>Investigator: Hanna<br /></b> <br/>
+<br/>
+<b>Comment:</b> Because we just used the Long-, Middle-, Short- and SEG-Linker for the small linker library, we decided also to perform the N-terminal fusion to VP2/3 with the GSAT linker. <br/>
+Yesterday, VP2/3 and VP2/3_Capins were cloned to the C-terminus of the GSAT linker. Ligation was performed over night.
+New plasmids: pSB1C3_GSAT_VP2/3 and pSB1C3_GSAT_VP2/3_Capins <br/>
+Trafo was performed according to standard protocol: XL1b cells, 2 µL DNA. Plates will be stored over night @ 37°C room. <br/>
+<b>To do tomorrow:</b> Picking clones.
+====<p style="font-size:15px; background-color:#66bbFF;"><b>Colony-PCR of pSB1C3_CD</b></p>====
+<b>Investigator: Bea</b>
+<br/>
+<p style="color:#66bbff;"><i>Comment</i>: Since the colony PCR of the last try did not work exactly the way we expected it, another colony PCR approach with new primers was conducted.</p>
+<br />
+<b>The protocol:</b>
+<ul>
+<li>Forward primer used: iGEM RFC25 </li>
+<li>Reverse primer used: iGEM RFC25 </li>
+<li>Cells: XL1-Blue</li>
+</ul>
+<br />
+<b>A mastermix for 37 samples was prepared for using it in this approach and the colony PCR for the loop insertion motif Z34C.</b><br/>
+<ul>
+<li>10x Taq Buffer: 1.0 µL</li>
+<li>MgCl<sub>2</sub> (50 mM): 0.1 µL</li>
+<li>dNTPs: 0.2 µL</li>
+<li>Primer iGEM RFC25 forward: 0.2 µL</li>
+<li>Primer iGEM RFC25 reverse: 0.2 µL</li>
+<li>Taq polymerase: 1.4 µL</li>
+<li>H<sub>2</sub>O: 231 µL</li>
+<li><b>Total volume for each reaction: 10 µL</b></li>
+</ul>
+<br />
+<b>PCR programm used:</b>
+<br>
+<br />
+Corresponding inoculated cells put in the 37°C room over night at 21:00.
+====<p style="font-size:15px; background-color:#66bbFF;"><b>BioBrick Assembly of ITR_phTERT_betaglobin_mVenus to hGH_rightITR</b></p>====
+<b>Investigator: Bea</b>
+<br/>
+<p style="color:#66bbff;"><i>Comment</i>: For assembling the last BioBricks together in order to obtain the fully assembled vectorplasmid which contains the tumorspecific promoter phTERT and the GOI mVenus.</p>
+<br />
+<b>Digestion of the constructs:</b>
+<ul>
+<li>P485 = pSB1C3_leftITR_phTERT_betaglobin_mVenus c=257 ng/µL</li>
+<li>P440 = pSB1C3_hGH_rightITR c=105 ng/µL</li>
+</ul>
+<br />
+{| border="1"
+| align="left" | '''Components''' ||align="left"| <b>v<sub>P485</sub> /µL</b> ||align="left"| <b>v<sub>P186</sub>/µL</b>
+|-
+| align="left" | DNA  ||align="left"| 4||align="left"| 14
+|-
+| align="left" | BSA (10x) ||align="left"| 2||align="left"| 2.5
+|-
+| align="left" | Buffer no. 4 (10x) ||align="left"| 2||align="left"| 2.5
+|-
+| align="left" | EcoRI ||align="left"| 1||align="left"| 1
+|-
+| align="left" | AgeI ||align="left"| 1||align="left"| 1
+|-
+| align="left" | H<sub>2</sub>O ||align="left"| 10||align="left"| 4
+|-
+| align="left" | '''Total volume''' ||align="left"| '''20''' ||align="left"| '''25'''
+|-
+|}
+<br />
+The expected fragments of the digested constructs were: The underlined fragments represent the fragments which correspond to the desired construct which can be used for ligation.
+<br />
+<ul>
+<li><b>P485:</b> <u>3911bp</u></li>
+<li><b>P186:</b> <u>657</u>, 2072bp</li>
+</ul>
+<br />
+<b>Loading plan: </b>
+ M    P485    P186
+<br />
+<b>Results:</b>
+[[Image: Freiburg10_pSB1C3_phTERT_mVenus_18_09_2010.jpg|thumb|center|400px]]
+<br />
+After gel extraction has been performed, the ligation was carried out.
+<br />
+<br />
+<b>Ligation:</b>
+<ul>
+<li>v<sub>=P485 </sub>=4,96µL </li>
+<li>v<sub>=P309 </sub>=3,04µL</li>
+</ul>
+The ligation mix was transformed into XL1-Blue cells and plated on agar plates containing chloramphenicol.
+<br />
+<br />
+<b>Next steps:</b>
+<br />
+Picking clones and perform Mini-Prep. After the Mini-Preps have been performed this construct needs to be sequenced and tested in cell culture.
+====<p style="font-size:15px; background-color:#66bbFF;"><b>Colony-PCR of ViralBricks</b></p>====
+<b>Investigator: Achim</b>
+<br/>
+<p style="color:#66bbff;"><i>Comment</i>: We decided to screen the new ViralBrick clones for the right insert via colony-pcr. This way we avoid unnecessary minipreps of clones containing religated vector or BLA. </p>
+<br />
+<b>The protocol:</b>
+<ul>
+<li>Forward primer used: iGEM RFC25 </li>
+<li>Reverse primer used: iGEM RFC25 </li>
+<li>Cells: XL1-Blue</li>
+</ul>
+<br />
+<b>A mastermix for 37 samples was prepared for using it in this approach and the colony PCR for the loop insertion motif Z34C.</b><br/>
+<ul>
+<li>10x Taq Buffer: 1.0 µL</li>
+<li>MgCl<sub>2</sub> (50 mM): 0.1 µL</li>
+<li>dNTPs: 0.2 µL</li>
+<li>Primer iGEM RFC25 forward: 0.2 µL</li>
+<li>Primer iGEM RFC25 reverse: 0.2 µL</li>
+<li>Taq polymerase: 1.4 µL</li>
+<li>H<sub>2</sub>O: 231 µL</li>
+<li><b>Total volume for each reaction: 10 µL</b></li>
+</ul>
+<br />
+<b>PCR programm used:</b>
+<br>
+<br />
+Corresponding inoculated cells put in the 37°C room over night at 21:00.
+<center>[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2 '''=> Go to Labjournal September part 2 (labday 124 - 135)''']</center><br>
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Team:Freiburg Bioware/NoteBook/Labjournal/September

From 2010.igem.org

Latest revision as of 21:34, 27 October 2010

Contents

September

107. labday 01.09.2010

Loop insertion BioBricks: Sequencing results

Design of primers for pAAV_RC

Sequencing results of pSB1C3_Zegfr:1907_Linker

Trafo evalutaion and Inoculation

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

Transfection, Seeding AAV293 and HT1080

Cloning of ZEGFR:1907_Middle-Linker,ZEGFR:1907_Short-Linker, ZEGFR:1907_SEG-Linker and ZEGFR:1907_Long-Linker into pCerulean

108. labday 02.09.2010

Sequencing of loop insertions BioBricks: 2nd round

Mini-preps of pSB1C3_SV40 and pCerulean_VP1_NLS

Mini-Prep and test digestion of pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker

Midi-Prep of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1 & pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1

Mini-Prep and test digestion of the CD biobricks

109. labday 03.09.2010

Miniprep and test digestion of pSB1C3_lITR_pTERT_ßglobin_mVenus_hGH_rITR

Sequencing results

>Cloning of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

Harvest viral particles, Seeding HT1080 cells

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

Cloning of pSB1C3_leftITR_pTert and pSB1C3_mGMK with pSB1C3_leftITR_CMV

Sequence analysis of pCerulean_VP1up

pCerulean_VP1up_NLS_Targeting molecule

3rd Repetition of Mini-Preps and test digestion for Loop insertion BioBricks

Cloning of Cap into pAAV_RC_ins-rep: preparation for SDM

110. labday 04.09.2010

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273): Sequencing results of P381 and P385

Harvest viral particles, Transduction

Repetition of Hybridisation and Cloning for Loop insertion BioBricks

Repetition: Prepatation for SDM: cloning of Cap into pAAV

Ligation and trafo of the modified Cap insert , the Viral Bricks 1, 11, 12, 13 and 14

Preparation of PBS Buffer

111. labday 05.09.2010

Mini-prep of glycerol stock pMA_RepCap Vector_SDM_InsPvuII clone 1 (B182)

>Picking clones of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

112. labday 06.09.2010

Minipreps & Test PCR of pTert Biobrick assembly

Minipreps & Test PCR of Loop inserts 1, 11, 12 ,13, 14

Mini-Preps and test digestion of Linker BioBricks

Production of Ampicilin and Chloramphenicol

Mini-prep and test digestion of pAAV_RC-CapIns_prepSDM

Sent for sequencing

113. labday 07.09.2010

Sequencing results of new loop insertion ligations

Repetition of CD biobrick production

Test digestion of pCerulean_VP1up_NLS_mVenus and pCerulean_VP1up_NLS_Affibody

Reftrafo of pSB1A2_EGFP_HQ from distribution plate (Bba_I719015 plate 1 well 15P = T7GFPmut3bHQ)

Design and Ordering of GFP-Oligos

Sequence analysis of pAAV_RC_CapIns_prepSDM

Cloning of Cap into pAAV_RC_1.2 SDM SalI

Repetition of Mini-Preps and test digestion of Linker BioBricks from 2nd ligation

Preparation for Midi-Preps of pAAV_mVenus, pHelper and pAAV_RC

Sequencing

114. labday 08.09.2010

Sequencing results of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

Repetition of PCR of SV40 terminator

PCR of P40

Midi-Prep

Seeding HT1080, Passaging AAV293 and Transfection

Checking fractions of Äkta run

115. labday 09.09.2010

Picking clones of leftITR_hTERT_beta-globin and leftITR_CMV_beta-globin biobricks

new ligation try and transformation of CD-biobrick

Repetition of ViralBrick Ligations: 453 Z34C, 587 Z34C, 587 KO Z34C, 587 KO Z34C Spacer

Midiprep of pHelper, pAAV_RC and pAAV_iGEM_mVenus (B34)

Miniprep of pAAV_RC_CapIns_noRep and pSB1A2_EGFP_HQ

PCR of VP2/VP3

Sequencing results of pCerulean_VP1up_NLS_Affibody/mVenus/Histag

Biobrick production of leftITR-phTERT-beta-globin and leftITR_pCMV-beta-globin

Transfection, Harvest viral particles and Transduction

Competent XL1-B cells

Checking PCR amplified virus particles

Quickchange site directed mutagenesis of pAAV_RC_CapIns_prepSDM

116. labday 10.09.2010

Trafo evaluation of CD-biobrick plate

Cloning of Z_EGFR:1907_Middle-Linker,Z_EGFR:1907_Short-Linker, Z_EGFR:1907_SEG-Linker and Z_EGFR:1907_Long-Linker into pCerulean

BioBrick ^TM assembly of pSB1C3_leftITR_promoter_betaglobin to several GOIs

3^rdrepetition of SV40 terminator (hopefully the last one ;-))