Team:Freiburg Bioware/NoteBook/Labjournal/September2

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August Part 2 (labday 99 - 106)

September

September Part 2 (labday 124 - xxx)

September

124. labday 19.09.2010

Continuation with Colony-PCR of pSB1C3_CD and ViralBrick motif Z34C

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.

Loading plan:
upper lanes

M   CD1  CD2  CD3  CD4  CD5  CD6  CD7  CD8  CD9  CD10   +ctrl(CFP) +ctrl(BAP) -ctrl(pAAV_MCS)   11Q1  12Q1  13Q1 13Q2  M

lower lanes

M   11T41  11T42  11T43  11T44    12T41  12T42  12T43  12T44    13T41  13T42  13T43  13T44    14T41  14T42 14T43  14T44  M

Expected sizes of the PCR products are:

Cytosine deaminase (CD) = 1300bp
Z34C loops insertion motif = 220bp
Positive control 1 = CFP = 750bp
Positive control 2 = BAP_587 = 170bp

Results:
The Colony PCR of the Cytosine Deaminase (CD) did not work out. The detectable bands at around 700 bp correspond to CFP. Therefore, BioBrick production of the CD must be repeated. The following three bands represent the controls. Three different controls were used. The first corresponds to pSB1C3_CFP, the second to BAP_587 and third control is the negative control. Unfortunately now bands can be detected in the postive controls. A faint band can be seen in the negative control which is the only lane in which no band should be detectable.
The following bands after the three controls correspond to the approaches of the Z34C Viralbrick production. Some bands in the lower and the upper lanes show positive results. The corresponding inoculated overnight culture were centrifuged and prepared in order to perform a Mini-Prep tomorrow.

Freiburg10 ColonyPCR Results 19.09.2010.jpg

Comment: hmm, seems that the CD-assambly didn't work at all.. Should i try it again or are we gonna skip it for good due to other priorities?(kira)
No, we cannot skip it. Can we change something else?? Any ideas what we can alter in the protocol? Because problem is that we are still waiting for the tk... so we should aswell focus on the other prodrug activating enzmye!! (Bea)

Testtransduction of the final modified capsid coding constructs

Investigator: Adrian

Aim of the experiment:
During the project 22 silent nucleotide exchange mutations were introduced into the capsid coding construct to make it compatible with the RFC standards and to have single cutting restriction enzymes flanking the 453 and the 587 loop sequence. Two point mutations had to be dismissed, because either a first test transduction showed that the construct was not working anymore or the insertion of the synthesized gene posed serious problems, because the restriction enzyme did not work.
Finally, we have a construct that is shown to produce infectious particles comparable to the current AAV systems and carries 20 point mutations. Now we can announce that the Adeno-associated Virus is compatible to the RFC standard and the idea to replace the loop sequences via ViralBricks works!

Impression from the moment:

The construct with the insertion of the Rep and the Cap synthesis worked! Reintroduction of the KpnI restriction site recovered the functionality of the viral genome!

Miniprep of serveral constructs

Investigator: Stefan

Glycerol stocks were prepared:

B430 = pCerulean_ZEGFR:1907_Longlinker_VP2/3
B431 = pCerulean_CFP_Middlelinker_VP2/3_insCap
B432 = pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap

Mini-Prep was performed according to the standard protocol

P527 = pCerulean_ZEGFR:1907_Longlinker_VP2/3 c = 230,61 ng/µl
P528 = pCerulean_CFP_Middlelinker_VP2/3_insCap c = 290,81 ng/µl
P529 = pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap c = 269,39 ng/µl

Several other constructs were preped, but need to be confirmed before being added to plasmid/glycerol stock.

125. labday 20.09.2010

Picking the clones and preparing the over-night culture

Investigator: Kira

VP2/3_Gsat-linker as well as VP2/3_cap_Gsat-linker plates contain colonies, thus 3 clones from each plate were inoculated into 5 ml DYT+ 5ul Chloramphenicol and incubated @ 37 C

Minipreps and Sequencing of ViralBrick clones

Investigator: Achim

The following clones were prepped:

Quickligation:
- 11.1, c = 85,41 ng/µl
- 13.1, c = 96,93 ng/µl
T4-Ligation:
- 11.4, c = 76,42 ng/µl
- 12.1, c = 80,36 ng/µl
- 12.4, c = 85,43 ng/µl
- 13.1, c = 90,06 ng/µl
- 13.2, c = 80,87 ng/µl
- 13.4, c = 76,62 ng/µl
- 14.1, c = 76,50 ng/µl
- 14.2, c = 98,52 ng/µl

Clones 11.4, 12.1, 13.2, 14.2 (T4) were sent for sequencing.

Cloning of pCerulean_Affibody_VP2/3 and pSB1C3_Affibody_middlelinker_GFP_his

Investigator: Jessica

Comment:Affibody_VP2/3 will be cloned in pCerulean and Affibody_middlelinker will be cloned together with GFP_His in pSB1C3

P407= pCerulean_CFP_middlelinker c=482,76 ng/µl
P516= pSB1C3_ZEGFR:1907_VP2/3 clone 1 c= 191,6 ng/µl
P290= pSB1C3_Affibody_Middlelinker clone 1 c= 227,4 ng/µl
P518= pSB1C3_GFP_RFC25_upper band clone 1 c= 138,3 ng/µl
P520= pSB1C3_GFP_RFC25_lower band clone 1 c= 141,6 ng/µl

Digestion:

components	P407	P516	P290	P518	P520
DNA	3,5	10	7	10	10
BSA (10x)	2	2	2	2	2
Buffer 4 (10x)	2	2	2	2	2
EcoRI	0,8	0,8	-	-	-
AgeI	0,8	0,8	0,8	-	-
NgoMIV	-	-	-	0,8	0,8
SpeI	-	-	0,8	0,8	0,8
H₂O	10,9	4,4	7,4	4,4	4,4
Total volume	20	20	20	20	20

1,0 g Agarose,100 ml TAE (1%), 6 µl GELRED , at 120 Volt

P516, P518, P520 will be repeated tomorrow (no DNA after gel-ex)

Gelextraction:

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

P407 c= 6,5 ng/µl
P516 c= - ng/µl
P290 c= 7,3 ng/µl
P518 c= - ng/µl
P520 c= - ng/µl

stands in 4°C room for comtinuation tomorrow

Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

Investigator: Anissa
Digestion of the constructs:

P320 = pSB1C3_001_RC_insrepcap_KpnI_back c=408 ng/µL
P320 = pSB1C3_001_RC_insrepcap_KpnI_back c=408 ng/µL
P180 = pSB1C3_pTAV2 (P5)clone 1 c=108,8 ng/µL
P184 = pSB1C3_pAAV_RC (P5TATAless)clone 3 c=176,9 ng/µL

Components	v_{P320 upstream} /µL	v_{P180 upstream}/µL	v_{P320 downsteam} /µL	v_{P184 downsteam}/µL
DNA	2,5	13,8	2,5	8,5
BSA (10x)	1,5	2	1,5	1,5
Buffer no. 4 (10x)	1,5	2	1,5	1,5
Enzyme 1	XbaI 1	SpeI 1	SpeI 1	XbaI 1
Enzyme 2	EcoRI 1	EcoRI 1	PstI 1	PstI 1
H₂O	7,5	0,2	7,5	1,5
Total volume	15	20	15	15

Loading plan:

M    P320(upstream)    P320(downstream)    P180    P184

Results:

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

Ligation:

v_{=P320_up}=7,37µL
v_{=P180_up}=0,63µL
v_{=P320_down}=7µL
v_{=P184_down}=1µL

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

Next steps:
Picking clones and perform Mini-Prep.

Test digestion of VP2-N-terminal fusion constructs

Investigator: Hanna

Comment: On Satureday a colony PCR was performed of:
1. pCerulean_Zegfr:1907_ShortLinker_VP2/3 (P530)
2. pCerulean_Zegfr:1907_SEG_VP2/3 (P531)
3. pCerulean_CFP_MiddleLinker_VP2/3 (P532)
4. pCerulean_6xHis_MiddleLinker_VP2/3 (P533)
5. pCerulean_6xHis_MiddleLinker_VP2/3_insCap (P534)
6. pCerulean_Zegfr:1907_MiddleLinker_VP2/3_insCap (P535)

Because all constructs (4 clones of each approach), including the negative control (pAAV_RFC25), showed positive results, one test digestion of each construct was performed.

Digestion

components	Components Master Mix /µl
BSA (10x)	7
Buffer 4 (10x)	7
EcoRI	3.5
SpeI	3.5
H₂O	35

--> 2 µL DNA + 8 µL Master Mix

Incubation: 1.5 h

Agarose-Gel:

0.4 g Agarose, 50 mL TAE (0.8 %), 3 µL GELRED, at 100 Volt, running time: 50 minutes

Sample	Sample/µl]	Loading dye (6x)/µl	Expected size 1	Expected size 2
P 530	10 µl	2 µl	2710 bp	3937 bp
P 531	10 µl	2 µl	2710 bp	3937 bp
P 532	10 µl	2 µl	2710 bp	3937 bp
P 533	10 µl	2 µl	2710 bp	3937 bp
P 534	10 µl	2 µl	2710 bp	3937 bp
P 535	10 µl	2 µl	2710 bp	3937 bp

Marker: GeneRuler ladder mix

	Marker /µL	Sample P530 /µl	Sample P531 /µl	Sample P532 /µl	Sample P533 /µl	Sample P534 /µl	Sample P535 /µl
Lane	4	12	12	12	12	12	12

Test Digestion N-terminal VP2 Fusion

Comment: Test digestion delivered positive results: Expected fragment size after VP2/3 fusion to Zegfr:1907_Linker: 2180 bp - compared to failed VP2/3 insertion: ~ 220 bp.
Expected fragment size after VP2/3 fusion to CFP_MiddleLinker: 2710 bp - compared to failed VP2/3 insertion: ~ 760 bp.
pCerulean_ZEGFR:1907_Longlinker_VP2/3
pCerulean_CFP_Middlelinker_VP2/3_insCap
pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap
pCerulean_ZEGFR:1907_Shortlinker_VP2/3
pCerulean_ZEGFR:1907_SEG_VP2/3
pCerulean_CFP_MiddleLinker_VP2/3
pCerulean_6xHis_MiddleLinker_VP2/3
pCerulean_6xHis_MiddleLinker_VP2/3_insCap
pCerulean_ZEGFR:1907_MiddleLinker_VP2/3_insCap
will be sent for sequencing to GATC.

BioBrick production: PCR of pAAV_RC_InsRepCap_KpnIback_VP1-ko and VP2-ko

Investigator: Stefan

DNA samples were diluted 1:1000

P455 = pAAV_RC_1.2 SDM SalI
P463 = pAAV_RC_CapIns_prepSDM clone 3

PCR progam:

Ingredients	Volume P455 / µl	Volume P463 / µl
5X Phusion HF buffer	10	10
10 mM dNTP mix	1	1
forward primer: O93	2,5	2,5
reverse primer: O115	2,5	2,5
DNA Template	3	3
DMSO	2	2
Phusion Polymerase	0,5	0,5
H₂O	28,5	28,5
Total volume	50	50

PCR program:

Cycles	Temperature / °C	Time / s
1	98	60
2 ( step 2-4: 8x)	98	15
3	59	25
4	72	75
5 (step 5-6: 17x)	98	15
6	72	85
7	72	300
Hold	4	4

Gel:
0,5 g agarose (1%), 50 ml TAE, 3µl GELRED running time: 45 minutes at 110V

Gel-Extraction:
Gel-extraction was performed according to standard protocol. The complete amount eluted was used for PCR digestion.

Digestion of PCR product:

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

Digestion was performed at 37 °C for 2 hours.

PCR-Purification:
Gel-extraction was performed according to standard protocol.

c (P455) = 13,5 ng/µl
c (P463) = ng/µl

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

Components	vector Volume/µL
DNA	3
BSA (10x)	2
Buffer no. 4 (10x)	2
EcoRI	1
SpeI	1
H₂O	11
Total volume	20

Digestion was performed at 37 °C for 2 hours.

Gel:
0,5 g agarose (1%), 50 ml TAE, 3µl GELRED running time: 50 minutes at 120V

Gel-Extraction:
Gel-extraction was performed according to standard protocol.
c(P320) = 0,8 ng/µl

Ligation:

1 µl T4 DNA ligase
1 µl 10x Buffer
8 µl DNA mix (vector + insert)

Amounts of DNA used:

c(P320) = 7,73 µl
c(P455) = 0,27 µl

c(P320) = 7,19 µl
c(P463) = 0,81 µl

Incubation time: 45 minutes at room temperature.

Transformation:
Transformation was performed according to standard protocol using XL1b cells and Chloramphenicol as antibiotic.

2x repetition of CD biobrick

Investigator: Kira

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 (1:100 dil)	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
10x	98°C	15"
	58°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. 6 h

1% agarose gel

According to the gel results, PCR was repeated twice.. But both tries revealed the same unexpected results. Insert from the last cloning procedure was found and used for the ligation and further transformation.

Ligation
T4 ligase was used
1 ul T4 Buffer
1 ul T4 Ligase
8 ul (0,9 ul vector+ 7,1 ul insert) DNA-mix

incubation @ RT for 45 min

Transformation was performed according to the standard protocol and the cells were spread on the agar plate containing Chloramphenicol.

126. labday 21.09.2010

Trafo evalutation of CD biobrick plate

Investigator: Kira

the agar plate was checked at approx. 4 pm and there were no colonies visible. Taking in account that the plate was incubated from 10.30 pm till 8 am @ 37C due to the electricity failure, the plate was put back @ 37C at 4.30 pm

Comment: the plate was picked this morning by a lab member and put in the coldroom

Continuation of cloning of pCerulean_Affibody_VP2/3 and pSB1C3_Affibody_middlelinker_GFP_his

Investigator: Jessica

Comment:Affibody_VP2/3 will be cloned in pCerulean and Affibody_middlelinker will be cloned together with GFP_His in pSB1C3

P407= pCerulean_CFP_middlelinker c=482,76 ng/µl
P516= pSB1C3_ZEGFR:1907_VP2/3 clone 1 c= 191,6 ng/µl
P290= pSB1C3_Affibody_Middlelinker clone 1 c= 227,4 ng/µl
P518= pSB1C3_GFP_RFC25_upper band clone 1 c= 138,3 ng/µl
P520= pSB1C3_GFP_RFC25_lower band clone 1 c= 141,6 ng/µl

Digestion:

components	P516	P518	P520
DNA	10	10	10
BSA (10x)	2	2	2
Buffer 4 (10x)	2	2	2
EcoRI	0,8	-	-
AgeI	0,8	-	-
MscI	0,8	-	-
NgoMIV	-	0,8	0,8
SpeI	-	0,8	0,8
H₂O	3,6	4,4	4,4
Total volume	20	20	20

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 120 Volt

Gelextraction:

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

P516= c= ng/µl
P518= c= ng/µl
P520= c= ng/µl

T4 Ligation:

The Ligation was performed as following:

Vector Volume: µl
Insert Volume: 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 (XL1blue). The cells were plated on a agar plate with Kana/Cm

Sequenzing result of pSB1C3_EGFP_His upper and (!) lower band

Investigator: Jessica
1. pSB1C3_EGFP_His suffix:

2. pSB1C3_EGFP_His prefix:

results look in both plasmids and are used for cloning of pSB1C3_Affibody_middlelinker_GFP_His

Midi-Prep of pHelper & pSB1C3_leftITR_CMV_beta-globin_mVenus_hGH_rightITR

Investigators: Chris W.

Midi-Preps of pHelper=P540 and pSB1C3_leftITR_CMV_beta-globin_mVenus_hGH_rightITR=P541

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

plasmid-no.	P540	P541
concentration (ng/µl)	480,45	1264,20

New aliquots (60µl) of XL1blue can be used ! (Jessica)

127. labday 22.09.2010

Sequencing results of ViralBrick ligations using dephosphorylated vector

Investigator: Achim

Sequencing confirmed a correct sequence for clone 11.4 (453 Z34C). Clone 12.1 (587 Z34C)has several missing bases at the 3' end of the insert, clone 13.2 has an a -> c mutation and a c insertion in the insert (although the a->c might be a sequencing error.). Clone 14.2 contained a c deletion.

We have several new approaches:

-Correction of the mutations via PCR: We'll use the hybridisation oligo for the side of the insert containing the mutations, the rfc 25 primer on the other side. The amplified insert will be ligated into dephosphorized vector.

-As a backup, SDM-primers will be ordered and should be available next week.

-Also, a new colony pcr with several clones from the last ligation plates will be carried out.

-clone 13.4 (already prepped) will be sent for sequencing

Cloning pSB1C3_Viral Brick 587KO-empty (B274/P542) into pSB1C3_VP2/3_Capins (B438/P514)

Investigator Patrick
After an over night digestion with BamHI and PvuII at 37°C ....

My dear friend - what do you mean with "..."?? overnight digest at which temperature?? Did you look it up whether the enzymes show star activity at longer digestions or not?? ;-)

The Gelextraction with P514 was performed according to the standard protocol yielding 16,9 ng/ml.
The P542 insert should be 48 bp long and therefore was extracted with a QIAGEN kit able to extract even very small DNA fragments (40 bp) (QiaEX II Gel Extraction Kit (150)).

Expected size of the fragments:
P542: 2112 & 48 bp
P514: 3956 & 48 bp

The 48 bp band of P514 can't be seen clearly on the picture due to the bad resolution.

Ligation with T4 DNA ligase: 1 µl buffer (10x), 1 µl T4 DNA ligase, 7 µl Vector, 1 µl Insert, 70 minutes.

The transformation with XL1B was performed according to the standard protocol and plated: pSB1C3_VP2/3_Capins_587KO-empty

Cellculture: Serum-free cells

Investigator Patrick
We want our virus production to be serum-free to simplify the following purification.
15 ml cell-medium, 25% serum-free: 3,75 ml serum-free DMEM + 11,25 ml DMEM
15 ml cell-medium, 50% serum-free: 7,5 ml serum-free DMEM + 7,5 DMEM

PCR of P40

Investigator: Jessica
DNA sample P432 was diluted 1:1000

Ingredients	Volume / µl
5X Phusion HF buffer	10
10 mM dNTP mix	1
forward primer: O152	2,5
reverse primer: O188	2,5
DNA Template	3,0
DMSO	-
Phusion Polymerase	0,5
H₂O	31
Total volume	50,5

PCR program:

Cycles	Temperature / °C	Time / s
	98	60
8x	98	15
	61	25
	72	8
17x	98	15
	69	25
	72	8
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:

Digestion of PCR product:

Components	PCR products Volume/µL
DNA	40
BSA (10x)	6
Buffer no. 4 (10x)	6
PstI	2
XbaI	2
H₂O	4
Total volume	60

Digestion was performed at 37 °C for 2 hours.

PCR-Purification:

c (P432) = 31,8ng/µl

Gel-Extraction:
Gel-extraction was performed according to standard protocol.
c(P51.2) = 3,7ng/µl

Ligation:

1 µl T4 DNA ligase
1 µl 10x Buffer
8 µl DNA mix (vector + insert)

Amounts of DNA used:

(P51.2) = 7,74 µl
(P432) = 0,26µl

Incubation time: 45 minutes at room temperature.

Transformation:
Transformation was performed according to standard protocol using XL1b cells and Chloramphenicol as antibiotic.

Mini-Prep and test digestion of pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko

Investigator: Stefan

Comment:

Glycerol stocks were prepared:

B463 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 1
B464 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 2

Mini-Prep was performed according to standard protocol:

P548 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 1 c = 435,82 ng/µl
P549 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko_P5tataless clone 2 c = 409,21 ng/µl

Test digestion:

Results:
500px

Cloning of CMV promotor into pSB1C3_VP123_inscap

Investigator: Anna

Vector: name: pSB1C3_VP123_inscap P489
Insert: name: pSB1C3_CMV P193
new vector name: pSB1C3_CMV_VP123_inscap P
buffer used: 4
DNA concentration (vector): 350,0 ng/µl ; DNA concentration (insert): 333,5 µg/µl

components	volume of pSB1C3_VP123_inscap /µl	volume of pSB1C3_CMV /µl
DNA	3	4,5
BSA (10x)	1,5	1,5
Buffer 4 (10x)	1,5	1,5
Enzyme I	EcoI HF 1	EcoI HF 1
Enzyme II	XbaI 1	SpeI 1
H2O	7	5,5
Total volume (e.g. 15,20,25,30 µl)	15	15

0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time:45

Loading plan for agarose gel:
Loading dye with SDS (6X), 3 µl
Marker used: GeneRuler ladder mix (Fermentas), 4 µl

	Marker	Sample 489, 18µl	Sample 193, 18µl
Lane	1	3	5
Fragment size		4404 bp	681 bp

Gel extraction:

	pSB1C3_VP123_inscap_cut	CMV
DNA-concentration [ng/µl]	14,09	32,61

T4 Ligation:
Volume vector: 3,86 µl
Volume insert: 4,14 µl

Transformation:

XL1B cells were used.

Sequencing of Zegfr:1907-Linker Library, His-Tag and imaging appoaches

Investigator: Hanna

Comment: After several problems were managed (cloning difficulties, colony PCR troubles, interchanges of samples,...), sequences of the final constructs needed to be verified in order to test the constructs in cell culture.

Sample gallery
pCerulean_CFP_MiddleLinker_ VP2/3_InsCap (P528) forward	pCerulean_CFP_MiddleLinker_ VP2/3_InsCap (P528) reverse
pCerulean_ZEGFR:1907_ShortLinker_ VP2/3_InsCap (P527) forward	pCerulean_ZEGFR:1907_ShortLinker_ VP2/3_InsCap (P527) reverse
pCerulean_6xHis_MiddleLinker_ VP2/3 (P533) forward	pCerulean_6xHis_MiddleLinker_ VP2/3 (P533) reverse
pCerulean_6xHis_MiddleLinker_ VP2/3_insCap (P534) forward	pCerulean_6xHis_MiddleLinker_ VP2/3_insCap (P534) reverse
pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_insCap (P535) forward	pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3_insCap (P535) reverse
pCerulean_ZEGFR:1907_SEG_ VP2/3_insCap (P502) reverse	pCerulean_ZEGFR:1907_LongLinker_ VP2/3_insCap (P503) reverse
pCerulean_ZEGFR:1907_MiddleLinker_ VP2/3 (P504) reverse

Comment: Sequences looked well. The 6 samples with VP2/3_insCap will be tested today in cell culture.

Mass Midi-Prep

Investigator: Chris W.

Midi-Prep of:

pSB1C3_001_RC_InsRepCap_KpnIback_P5tataless clone 1 =P544
pSB1C3_001_RC_InsRepCap_KpnIback_P5tataless clone 2 =P545
pSB1C3_001_P5_RC_InsRepCap_KpnIback clone 1 =P546
pSB1C3_001_P5_RC_InsRepCap_KpnIback clone 2 =P547
pHelper =P550
pHelper =P551
pSB1C3_litr_pTert_ßglobin_mvenus_hgh_ritr clone 1 =P552
pAAV_RC_RepCapIns_SDMKpnl_VP1ko clone 1 =P553
pAAV_RC_RepCapIns_SDMKpnl_VP2ko clone 1 =P554
pAAV_RC_RepCapIns_SDMKpnl_VP2ko clone 1 =P555
pCerulean_ZEGFR:1907_SEG_VP2/3_CapIns clone 3 =P556
pCerulean_ZEGFP:1907_LongLinker_VP2/3_CapIns clone 2 =P557
pCerulean_CFP_Middlelinker_VP2/3_insCap =P558
pCerulean_ZEGFR:1907_Shortlinker_VP2/3_insCap =P559
pCerulean_6xHis_MiddleLinker_VP2/3_insCap =P560
pCerulean_ZEGFR:1907_MiddleLinker_VP2/3_insCap =P561
pCerlulean_ZEGFR:1907_Shortlinker_VP2/3_insCap clone 4.8 =P562

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

plasmid-no.	P544	P545	P546	P547	P550	P551	P552	P553	P554	P555	P556	P557	P558	P559	P560	P561	P562
concentration (ng/µl)	1851,91	1822,3	1783,48	2625,57	1524,63	3333,48	2641,57	2165,22	1818,23	2318,58	3255,81	2818,15	2761,32	3289,23	3134,26	3725,27	1965,28

RNA isolation and cDNA synthesis

Investigator Kira

Motivation: in order to figure out if our cell lines do express telomerase, cell pellets were used for RNA isolation.

RNeasy Kit was used for RNA isolation. The cells were harvested and pellets washed 2x with PBS. add RLT buffer
homogenize the lysate with syringe ans blunt needle
add 1 volume of 70% EtOH
centrifuge for 15s @ 10.000 rpm
add 700 ul Buffer RW1 and centrifuge again
add 500 ul Buffer RPE and centrifuge
add 500 ul Buffer RPE and centifuge for 2 min
place the column in 1.5 ml tube and add RNase free water (50 ul)

concentrations:
c(293)= 41, 68 ng/ul
c(a431) = 135, 48 ng/ul

Gel apparatus (chamber, tray and comb) for RNA purity gel has to be prepared a day ahead with : water, SDS and NaOH

cDNA synthesis

Investigator: Kira

The yielded DNA was used for cDNA synthesis.
QuantiTect Reverse Transcription (Qiagen) reaction was performed according to the manufacturer protocol. DNA elimination reaction mix:

components	a431/µl	293/µl
7xgDNA Wipeout Buffer	2	2
Template RNA	1,6	2
RNase-free H2O	10,4	10
Total volume	14	14

Reverse-transcription reaction mix:

components	1xreaction	MM
Quantiscript Reverse Transcriptase	1	3
5x Quantiscript RT buffer	4	12
RT primer Mix	1	3
DNA elimination reaction mix	14	-
Total volume	20	-

incubation for 15 min @ 42C
incubation for 3 min @ 95C

128. labday 23.09.2010

Colony PCR of new clones from the last ligations

Investigator Achim

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Contuniation: Cloning pSB1C3_Viral Brick 587KO-empty (B274/P542) into pSB1C3_VP2/3_Capins (B438/P514)

Investigator Patrick
3 Clones were picked in the morning and in each case inoculated 10 ml DYT. Clone 3 didnt grew fast enough. There were not enough bacteria in the eppi even after 8 ml were centrifuged. The mini-prep of clone 1 and 2 was performed according to the standrd protocol yielding the following concentrations:
Clone 1: 36,64 ng/µl
Clone 2: 7,8 ng/µl
Therefore only clone 1 was sent for sequencing (labelling: pat1, primer:VR2) and only this clone received a plasmid number: P569
No test-digestion was performed.
Because all medium was used to receive a sufficient amount of bacteria from clone 1 there was no cell suspension left to prepare a glycerol stock. Therefore again each clone was used to inoculated 10 ml DYT, following a mini-prep tomorrow.

Sequencing results:

Obviously there is something wrong with the backbone or the sequencing.

Mini-Prep and test digestion of pCerulean_Affibody_VP2/3, pSB1C3_Affibody_middlelinker_EGFP_His, pSB1C3_001_RC_InsRepCap_KpnIback_VP2-ko

Investigator: Jessica

Comment:

Glycerol stocks were prepared:

B465 = pCerulean_ZEGFR:1907_VP23 clone1
B466 = pCerulean_ZEGFR:1907_VP23 clone2
B467 = pSB1C3_ZEGFR:1907_middlelinker_EGFP_His clone1
B468 = pSB1C3_ZEGFR:1907_middlelinker_EGFP_His clone2
B469 = pSB1C3_001_RC_InsRepCap_KpnIbackVP2-KO clone1
B470 = pSB1C3_001_RC_InsRepCap_KpnIbackVP2-KO clone2

Mini-Prep was performed according to standard protocol:

P563 = pCerulean_ZEGFR:1907_VP23 clone1 = 227,4 ng/µl
P564 = pCerulean_ZEGFR:1907_VP23 clone2 = 337,2 ng/µl
P565 = pSB1C3_ZEGFR:1907_middlelinker_EGFP_His clone1 = 252,7 ng/µl
P566 = pSB1C3_ZEGFR:1907_middlelinker_EGFP_His clone2 = 209,5 ng/µl
P567 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-KO clone1 = 304,7 ng/µl
P568 = pSB1C3_001_RC_InsRepCap_KpnIback_VP1-KO clone2 = 383,1 ng/µl

Test digestion of

P563 = pCerulean_ZEGFR:1907_VP23 clone1

P564 = pCerulean_ZEGFR:1907_VP23 clone2

P565 = pSB1C3_ZEGFR:1907_middlelinker_EGFP_His clone1

P566 = pSB1C3_ZEGFR:1907_middlelinker_EGFP_His clone2

Components	P563-566 Volume/µL
DNA	2
BSA (10x)	1
Buffer no. 4 (10x)	1
PstI	0,5
XbaI	0,5
H₂O	5
Total volume	10

Results:

P563 and P566 was sent for sequencing with primer VF2 and VF2/VR2

PCR for 587 Z34C ViralBricks

Investigators: Achim, Anna

Plasmids used as template:

12_T4_1: c = 80,36 ng/µl
13_T4_2: c = 80,87 ng/µl
14_T4_2: c = 98,52 ng/µl

Primer used:

Template	Primer for	Primer rev
12_T4_1	RFC25	O146
13_T4_2	RFC25	O148
14_T4_2	O149	RFC25

Ingredients	Volume / µl
5X Phusion HF buffer	10
10 mM dNTP mix	1
forward primer: see above	2,5
reverse primer: see above	2,5
DNA Template (1:100 dilution)	1,0
DMSO	1,0
Phusion Polymerase	0,5
H₂O	31,5
Total volume	50

PCR program:

Cycles	Temperature / °C	Time / s
	98	60
	98	15
25x	68	25
	72	10
1x	72	300
Hold	4°C

Digestion of samples:

Restriction enzymes used: BamHI and PvuI.

Comment:

PCR of Vp1_ex, Vp2_ex and Vp3_ex and cloning into pSB1C3_001

Investigator: Anissa

DNA sample P72 (pkex_Vp1), P73(pkex_Vp2) and P74(pkex_Vp3) were diluted 1:100
used primers: O89 = Präfix_Vp1ex, O90= Präfix_Vp2ex, O91= Präfix_Vp2ex, O92= Suffix_Vp123ex, O120= Suffix_Vp123ex_reg

Ingredients	Volume / µl
5X Phusion HF buffer	10
10 mM dNTP mix	1
forward primer: O152	2,5
reverse primer: O188	2,5
DNA Template	2,0
DMSO	1
Phusion Polymerase	0,5
H₂O	30,5
Total volume	50

PCR program:

Cycles	Temperature / °C	Time / s
1x	98	60
25x	98	15
25x	60	25
25x	72	55
1x	72	5
Hold	4

Comment:The primer O120 was firstly given to the samples. It's a different primer for the Vp123_suffix, which binds to the regulatory-sequence. Because there was no knowledge, if the Vp_ex constucts have this sequence or not, O92 was also given to the samples.

Comment:Because gel-picture showed only DNA within the gel-bags, PCR was repeated.

Digestion of plasmid backbone:
Plasmid used: pSB1C3_001 (P320)
c (pSB1C3_001) = 408 ng/ µl

Components	vector Volume/µL
DNA	2,5
BSA (10x)	1,5
Buffer no. 4 (10x)	1,5
SpeI	1
NgomIV	1
H₂O	7,5
Total volume	15

incubation @ 37 C for approx. 1,5 h

Comment:Strangely, PCR-products did not run into the gel, altough the PCR was repeated, after first PCR had two reverse primers in the sample. This could be because of a complex with the polymerase.However, ligation will be continued with the DNA within the bags.

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Digestion of PCR product:

Components	PCR products Volume/µL
DNA	20
BSA (10x)	3
Buffer no. 4 (10x)	3
SpeI	1
NgomIV	1
H₂O	2
Total volume	30

Digestion was performed at 37 °C for 2 hours.

PCR-Purification:

was peformed according the standard-protocol.

c (P432) = ng/µl

Ligation:

1 µl T4 DNA ligase
1 µl 10x Buffer
8 µl DNA mix (vector + insert)

Amounts of DNA used:

(P51.2) = µl
(P432) = µl

Incubation time: 45 minutes at room temperature.

Transformation:
Transformation was performed according to standard protocol using XL1b cells and Chloramphenicol as antibiotic.

RNA Purity Gel

Investigator: Kira
10x FA buffer: 200mM MOPS, 50mM Sodium Acetat, 10mM EDTA, RNase free water
1x FA running buffer: 10x FA buffer, 37% Formamid, RNAase free water

Agarose gel: Agarose 0,6 g; 10x FA Buffer 5ml; RNAse free water 45ml; 37% Formamid 0,9 ml

hTERT PCR Reaction

Investigator: Kira

@Kira: Do you think that we can (suggestion?) use the A431 for detection of gene expression driven by phTERT promoter?? We should keep in mind that the transduction efficieny will not be that well ;-) (Bea)

Ingredients	Volume / µl
10x PCR buffer	2
10 mM dNTP mix	0,4
50mM MgCl2	0,6
Primer 1	0,5
Primer 2	0,5
cDNA template	1
Taq Polymerase	0,1
H₂O	14,9
Total volume	20

PCR program:

Cycles	Temperature / °C	Time
1x	94	3min
30x	94	45sec
	51	30
	72	1min 30sec
1x	72	10
Hold	4

1% agarose gel:

Evaluation: The agarose gel reveals that all samples show telomerase activity but in different levels.

Cloning of pBAD_Affibody_middlelinker_EGFP_His

Investigator: Jessica

Vector: name: pBAD_ OmpA His FluA LL Foki PGerrit
Insert: name: pSB1C3_ZEGFR:1907_middlelinker_EGFP_His P566
new vector name: pBAD_ZEGFR:1907_middlelinker_EGFP_His P
buffer used: 4
DNA concentration (vector): 97,0 ng/µl ; DNA concentration (insert): 209,5 µg/µl

components	volume of pBAD_ OmpA His FluA LL Foki /µl	volume of pSB1C3_ZEGFR:1907_middlelinker_EGFP_His /µl
DNA	10	6
BSA (10x)	2	2
Buffer 4 (10x)	2	2
Enzyme XbaI	0,8	0,8
Enzyme AgeI	0,8	0,8
H2O	4,4	8,4
Total volume (e.g. 15,20,25,30 µl)	20	20

0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt

Loading plan for agarose gel:
Loading dye with SDS (6X), 3 µl
Marker used: GeneRuler ladder mix (Fermentas), 6 µl

	Marker	Sample PGerrit, 20µl	Sample 566, 20µl
Lane	1	3	5
Fragment size		4047 bp	963 bp

Gel extraction:

	pBAD	Affibody_middlelinker_EGFP_His
DNA-concentration [ng/µl]	8,3	10,2

T4 Ligation:
Volume vector: 5,06 µl
Volume insert: 2,94 µl

Transformation:

XL1B cells were used.

Picking clones of pSB1C3_CMV_VP123_inscap

Investigator: Anna

Three clones were picked (10 ml DYT, 10 µl Cm).

Cloning of * into pSB1C3_CFP

Investigator: Chris W.

Vector: name: pSB1C3_CFP P51,2
Insert: name: email Hybrid Oligo (O184&O185)
new vector name: pSB1C3_CMV_email
buffer used: 4
DNA concentration (vector): 151,0 ng/µl ; DNA concentration (insert): 95,3 µg/µl

Interesting, where does the promoter come from the new annotared vector?? ;-)

components	volume of pSB1C3_CFP /µl	volume of email/µl
DNA	6	15
BSA (10x)	2	2
Buffer 4 (10x)	2	2
Enzyme I	NgoMIV 1	NgoMIV 1
Enzyme II	SpeI 1	SpeI 1
H2O	8,4	-
Total volume (e.g. 15,20,25,30 µl)	20	20

0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 120 Volt, running time:45

Loading plan for agarose gel:
Loading dye with SDS (6X), 4 µl
Marker used: GeneRuler ladder mix (Fermentas), 3 µl

	Marker	Sample Insert, 20µl	Sample Vector, 20µl
Lane	1	3	5
Fragment size		70 bp	2070 bp

Gel extraction:

	pSB1C3_CFP_cut	email
DNA-concentration [ng/µl]	7,8	27,4

T4 Ligation:
Volume vector: 8,74 µl
Volume insert: 0,26 µl

Transformation:

XL1B cells were used.

Repetition: Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

Investigator: Stefan
Digestion of the constructs:

P432 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl
P432 = pSB1C3_001_RC_insrepcap_KpnI_back c=344,5 ng/µl
P180 = pSB1C3_pTAV2 (P5)clone 1 c=108,8 ng/µl
P184 = pSB1C3_pAAV_RC (P5TATAless)clone 3 c=176,9 ng/µl

Components	V P432up /µL	VP180up /µL	V P432down /µL	V P184down /µL
DNA	4,5	14	4,5	11
BSA (10x)	2	2	2	2
Buffer no. 4 (10x)	2	2	2	2
Enzyme 1	XbaI 1	SpeI 1	SpeI 1	XbaI 1
Enzyme 2	EcoRI 1	EcoRI 1	PstI 1	PstI 1
H₂O	9,5	9,5	-	3
Total volume	20	20	20	20

Results:

Gel-Extraction: Gel-Ex was performed according to protocol. The following concentrations were measured:

432up c= 25,73 ng/µl
180up c= 9,57 ng/µl
432down c= 27,34 ng/µl
184down c= 17,80 ng/µl

Ligation:
The following DNA mixes were used for ligation:

432up V= 6,3 µl
180up V= 1,7 µl

432down V= 6,93 µl
184down V= 1,07 µl

Transformation:
The transformation was performed according to standard protocol using BL21 cells. They were plated on agar plates containing chloramphenicol and stored in 37°C room overnight.

129. labday 24.09.2010

Contuniation: Cloning pSB1C3_Viral Brick 587KO-empty (B274/P542) into pSB1C3_VP2/3_Capins (B438/P514)

Investigator Patrick

Team:Freiburg Bioware/NoteBook/Labjournal/September2

From 2010.igem.org

Contents

September

124. labday 19.09.2010

Continuation with Colony-PCR of pSB1C3_CD and ViralBrick motif Z34C

Testtransduction of the final modified capsid coding constructs

Miniprep of serveral constructs

125. labday 20.09.2010

Picking the clones and preparing the over-night culture

Minipreps and Sequencing of ViralBrick clones

Cloning of pCerulean_Affibody_VP2/3 and pSB1C3_Affibody_middlelinker_GFP_his

Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

Test digestion of VP2-N-terminal fusion constructs

BioBrick production: PCR of pAAV_RC_InsRepCap_KpnIback_VP1-ko and VP2-ko

2x repetition of CD biobrick

126. labday 21.09.2010

Trafo evalutation of CD biobrick plate

Continuation of cloning of pCerulean_Affibody_VP2/3 and pSB1C3_Affibody_middlelinker_GFP_his

Sequenzing result of pSB1C3_EGFP_His upper and (!) lower band

Midi-Prep of pHelper & pSB1C3_leftITR_CMV_beta-globin_mVenus_hGH_rightITR

New aliquots (60µl) of XL1blue can be used ! (Jessica)

127. labday 22.09.2010

Sequencing results of ViralBrick ligations using dephosphorylated vector

Cloning pSB1C3_Viral Brick 587KO-empty (B274/P542) into pSB1C3_VP2/3_Capins (B438/P514)

Cellculture: Serum-free cells

PCR of P40

Mini-Prep and test digestion of pSB1C3_001_RC_InsRepCap_KpnIback_VP1-ko

Cloning of CMV promotor into pSB1C3_VP123_inscap

Sequencing of Zegfr:1907-Linker Library, His-Tag and imaging appoaches

Mass Midi-Prep

RNA isolation and cDNA synthesis

cDNA synthesis

128. labday 23.09.2010

Colony PCR of new clones from the last ligations

Contuniation: Cloning pSB1C3_Viral Brick 587KO-empty (B274/P542) into pSB1C3_VP2/3_Capins (B438/P514)

Mini-Prep and test digestion of pCerulean_Affibody_VP2/3, pSB1C3_Affibody_middlelinker_EGFP_His, pSB1C3_001_RC_InsRepCap_KpnIback_VP2-ko

PCR for 587 Z34C ViralBricks

PCR of Vp1_ex, Vp2_ex and Vp3_ex and cloning into pSB1C3_001

RNA Purity Gel

hTERT PCR Reaction

Cloning of pBAD_Affibody_middlelinker_EGFP_His

Picking clones of pSB1C3_CMV_VP123_inscap

Cloning of ***** into pSB1C3_CFP

Repetition: Cloning of P5_TATAless downstream of pSB1C3_001_RC_insrepcap_KpnI_back and P5 upstream of pSB1C3_001_RC_insrepcap_KpnI_back

129. labday 24.09.2010

Contuniation: Cloning pSB1C3_Viral Brick 587KO-empty (B274/P542) into pSB1C3_VP2/3_Capins (B438/P514)

Cloning of * into pSB1C3_CFP