Team:Freiburg Bioware/NoteBook/Labjournal/July

• Because the previous attempt to replace the PSTI and NOTI restriction sites in the ITRs with the IGEM standard were unsucessful, new, longer primers were designed an ordered.
• a new pcr reaction with higher annealing temperatures was prepared:

PCR program:

• Mg concentration could be optimized
• Template okay? Template will be loaded on gel as well next time

Search for restriction sites in the sequence LQA/RGQA/RQA

Investigator: Volker

The idea to use a R588L mutation instead of the R588A that is published in the literature was discussed with the supervisers. It was proposed to search for other amino acid exchanges that alter the loop less drastically. For this reason the mutation N587Q was examined for possible restriction sites.

Search for restriction sites in the 585/588 KO loop sequence

Results sequencing alignment of GMK_TK30

Investigator: Bea

Result: The obtained sequencing results indicate that the DNA we received from Amor contains the GMK_TK30 fusion enzyme, but tk30 contains some restriction sites.

Sequence alignment of GMK_TK30

Sequence alignment of GMK_TK30

Sequence alignment of GMK_TK30

Sequence alignment of GMK_TK30

• To do (06.07.2010): Order primers for BioBrick Fusionenzyme and order genes of GMK and TK (wtTK, TK30, sr39????)
• remove iGEM restriction sites

51.Labortag 6.07.2010:

Harvesting AAVs from 4 10cm dishes, Transduction of 3x6er dishes

Investigators: Adrian, Anna, Bea, Kerstin

We want to investigate which of the following approaches yields a better transduction efficiency.

• Half of the transfected cells were exposed 3 cycles of thawing and freezing and then centrifuged (15 ml falcon, 2100G). The supernatant was transfered into a new 15 ml falcon and used for transduction.
• The other half was centrifuged at 300 G for 5min . The supernatant was transfered into another 15 ml falcon and the pellet was resuspended with 5 ml of DMEM. The content of these two falcons was also exposed to 3 cycles of thawing and freezing and then used for transduction.

• We have got 2 10 cm cellculture dishes with 10µg and 2 dishes with 20µg DNA used for transfection.

Approach with standart protocol (one dish with 10 and the other with 20µg Plasmids)(2100 G and 3 cycles of freezing and thawing)

Deviations from the standart protocol:

• The cells were centrifuged at 2100 G instead of 10.000 G
• The viruses were harvested after 44 hours
• 3 cycles of freezing and thawing

Search for restriction sites before and after the insertion sites 453 and 585/588

Investigator: Volker

Transduction of 3x6er dishes

Investigators: Adrian Bea

• used plasmids: 10µg, 20µg YFP
• we have 10µg and 20µg from standart protocol => SV
• we have 10µg and 20µg from standart protocol pellet => Pellet
• we have 10µg and 20µg from standart protocol suspension => Super
• amount of 500µl

1. Plate I(A is up)

2. Plate I(A is up)

3. Plate I(A is up)

Search for restriction sites around the 453 and 585/588 integration sites

Investigator: Volker

For this purpose a list of all commercially availible restriction enzymes was created that do not match in the ORFs of pAAV-RC.

Search for restriction sites in the sequence LQA/RGQA/RQA

Investigator: Volker

The idea to use a R588L mutation instead of the R588A that is published in the literature was discussed with the supervisers. It was proposed to search for other amino acid exchanges that alter the loop less drastically. For this reason the mutation N587Q was examined for possible restriction sites.

bla Bla bla

bla Bla bla

52.Labortag 07.07.2010:

Sequencing of GIO

Investigator: Hanna
Comments: Neither sequencing with forward nor with reverse primer (O29 + O30) worked. Therefore P40 (pAAV_iGEM_mVenus-YFP) was sent for sequencing once again.

• Name: Hanna P40
  
• V (P40) = 11 µL + 19 µL H2O
  
• Name of primer forward: Hanna 1 (O30)
  
• Name of primer reverse: Hanna 2 (O29)
  
• V(primer) = 3 µL + 27 µL H2O
  

Interview with Bild der Wissenschaft

• Adrian did a good job ;-)
• The article will be send to us next week
• For additional question, Jessica will be send an email

Transfection

Investigators: Adrian, Bea

• Transfection of AAV-293 cells with pAAV_iGEM-mVenus_YFP

New pcr run with new polymerase and different MgSO4 concentrations

• While this time the positive control was actually positive, no pcr product could be seen. The reasons are unclear. (Secondary structures, primer binding...)

Checking the results of the following sequencing reactions for the expression constructs from the DKFZ

Investigator: Volker

pKEX-VP1ex: sequence confirmed as described in [Name et al.,] with two pointmutations (ACG=>GCG) and (ATG=>CTG) to avoid the translation of VP2 and VP3.

pKEX-VP2ex: sequence confirmed as described in [Name et al.,] with one mutation (ATG=>CTG) to avoid the translation of VP3 and a second mutation (AAGACG=>CATATG) to switch to the main start codon ATG and to introduce a restriction site (which one??) before the start codon.

pKEX-VP3ex: sequence confirmed as described in [Name et al.,] with two silent pointmutations and a second mutation to introduce a restriction site before the start codon.

pKEX-Rep40ex: Sequence as expected until now, further sequencing reaction required with the primer: GATC_std_pTeSp-1

pKEX-Rep52ex: Point mutation directly at the beginning of the exon sequence, perhaps functionality in this context? At the end of the sequence some umcertainities. For the beginning further sequencing reaction required with the primer: GATC_std_pTeSp-1

pKEX-Rep68ex: One point mutation in the second condon that changes the coded amino acids from P to A, a second mutation (ATG=>GGG) at the beginning of the Rep40/Rep52 ORFs to avoid the translation of these sequences and a silent mutation. One further sequencing reaction is required with the primer: GATC_std_pcDNA1.1-RP.

pKEX-Rep78ex: One point mutation in the second condon that changes the coded amino acids from P to A, a second mutation (ATG=>GGG) at the beginning of the Rep40/Rep52 ORFs to avoid the translation of these sequences, a silent mutation and the same point mutation at the beginning of the exon as seen before in pKEX-Rep52ex.

53.Labortag 08.07.2010:

Final planing of the Cap modifications

Investigator: Volker

BbvCI
50|100|10|100 37°
File:Freiburg10 BbvC-I-cutsite 1 v1 000007.gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3:

KpnI
100|75|0|50 37°C + BSA
File:Freiburg10 Kpn-I-cutsite 1 v1 000021.gif.gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3:

SalI
10|100|100|100 37°C
File:Freiburg10 Sal-I-cutsite 1 v1 000016.gif.gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3:

AarI
|||
File:Freiburg10 .gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3:

ScaI
100|100|10|100 37°C
File:Freiburg10 Sca-I-cutsite 1 v1 00001.gif.gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3: 2

BamHI
75|100|100|100 37°C + BSA
File:Freiburg10 BamH-I-cutsite 1 v1 000025.gif.gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3:

PvuII
100|100|100|100 37°C
File:Freiburg10 Pvu-II-cutsite 1 v1 000013.gif.gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3: 1

EagI
10|25|100|10 37°C
File:Freiburg10 Eag-I-cutsite 1 v1 000021.gif.gif
Recognition sites in Rep/Cap ORFs:
Recognition sites in pAAV_RC:
Recognition site in pSB1C3: 2

54.Labortag 09.07.2010:

Investigators: Anissa, Kerstin, Anna

• New DMEM-Medium was prepared (10% FCS). Investigators: Bea, Patrick
• HT and 293 cells were split into 4 flasks, 2 flasks each. Investigator: Kira
• AAV stocks were prepared, put into the -80°C freezer and labeled with the date, name, AAV, the amount of DNA used for transfection and drop/resuspend to make clear whether the DNA was just pippeted dropwise to the 293 cells or gently mixed with the cells. The numbers on the 15 ml falcons refer to DNA amount and DNA administration, too. Investigator: Patrick

PCR of hgH

• PCR program: Unnamed

Agarose-Gel:

0,625 g Agarose, 50 ml TAE (1,25%), 3 µl GELRED (3-6µl), at 110 Volt, running time: 60 minutes

• Marker: GeneRuler ladder mix

Gelextraction

Gel measurement:

• DNA concentration (hgH): 19,83 ng/µl

Comments: PCR was succesful:-) to do: Ligation of PCR product into standard plasmid which we´ve received wihthin the DNA distrivution kit of iGEM-HQ .

Order oligos for GMK_TK30

Investigator: Bea
Oligos have been ordered at sigmaaldrich in order to insert prefix and suffix to fusionenzyme mGMK_TK30 in RFC25.

to do: Order gene tk30 and part of gene sr39

Quickchange for the PstI restriction sites in pAAV-RC

Investigators: Kira and Volker
Quick change, Gel and test digestion with PstI. (more precise informations will be added)
@Kira and Volker: did the Quickchange actually work? Could you please add the information onto the wiki!! Thx!

PCR program: PstI

• The PCR-products were digested with DpnI (1µl instead of 0.5 µl)
• aragose gel revealed for both samples very distinct bands

TEST DIGESTION

• agarose gel was performed with digested samples and revealed 3 distinct bands, while only 1-2 bands have been expected. Because of the density of the bands it might be that the digestion with DpnI did not work properly, thus additional 1 µl of DpnI was added to each sample. Incubation @ 37 C lasted 1 h.

55.Labortag 11.07.2010

Splitting of HT1080 cells

Investigator: Patrick
HT 1080 and HEK 293 cells were split.
@ Kira: fetch new trypsin if it is not sufficient for the next splitting !

56.Labortag 12.07.2010:

ITR BioBrick production

Investigator: Anna, Kerstin, Anissa

Practical Cloning:

• experiment date: 12.07.2010 ; time: 11.00
• plasmid: pSB1c3_CFP (p48)
  • Vector: name: pSB1c3 number:- production date: - origin: iGEM
  • Insert: name: pMA_CFP number:- production date: - origin: Sven
• new vector name: pSB1c3_CFP (p48)
• buffer used: 3 ; Restriction-enzymes used: Enzyme 1 (no. Lab:28) EcoRI ; Enzyme 2 (no.Lab:48) PstI ;Enzyme 3 (no.Lab:77) DpNI
• DNA concentration (vector): 25 µg/µl ; DNA concentration (insert): 436,8 µg/µl

Comments:Third enzyme was only used to digest template from vector pcr

Digestion

• Incubation: 1h

Agarose-Gel:

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED (3-6µl), at 110 Volt, running time:45 minutes

• Marker: GeneRuler ladder mix

Gelextraction

Gel measurement:

Ligation

TRAFO-EVALUATION

• Unfortunately the agar plate with p48, pSB1c3_CFP [XL1B, Cm, A.B.] does not contain any colonies. Thus, the plate was incubated @ 37 C for another 5 hours. Still no detectable colonies. This was double checked by Sven.

Seeding HT1080 + Passage

• Investigators: Adrian, Patrick
• 3* 6well dishes

• The amount of cells was doubled in first place and diluted in following steps (for further information ask patrick and adrian)

PCR of beta-globin:

• Investigator: Achim
• PCR program: BETA...(?)

Agarose-Gel:

0,625 g Agarose, 50 ml TAE (1,25%), 3 µl GELRED (3-6µl), at 115 Volt, running time: 30 minutes

• Marker: GeneRuler ladder mix

Gelextraction

Gel measurement:

• DNA concentration (hgH): 34,87 ng/µl

Comments: PCR was succesful:-)
to do: Ligation of PCR product into standard plasmid which we´ve received wihthin the DNA distrivution kit of iGEM-HQ .

Midi-Prep preparation pAAV_RC

Investigator: Bea

• 200mL LB-Medium and 200µl Amp
• inoculated with glycerol stock containing pAAV_RC
• 20:00 p.m.: put intp 37°C room on shaker

First steps in producing ITR BioBricks via Fancy Method

Investigators: Hanna, Chris W.

Digestion of pGA14_Cerulean-CFP

1. Digestion with EcoRI and NotI:

Incubation: 1.5 hours, 37°C

Gel run: 20 µL sample + 4 µL loading dye (6x); 8µL marker
Expected fragment size: ~ 2900 bp

Gel extraction:
Sample weight: 0.15 g
Gel extraction was performed following the standard protocol.

DNA concentration: 26.8 ng/µL

1. Digestion with XbaI and PstI:

Incubation: 1.5 hours, 37°C

Gel run: 20 µL sample + 4 µL loading dye (6x); 8µL marker
Expected fragment size: ~ 2900 bp

Gel extraction:
Sample weight: 0.17 g
Gel extraction was performed following the standard protocol.

DNA concentration: 22.9 ng/µL

Digestion of pAAV_MCS (P9) with AlwNI

Incubation: 1 hours + 20 minutes, 37°C

Gel run: 20 µL sample + 4 µL loading dye (6x); 8µL marker
Expected fragment sizes: 2982 bp = right ITR and 1674 bp = left ITR

Gel extraction:

• Sample: right ITR = 0.15 g
  
• Sample: left ITR = 0.12 g
  

Gel extraction was performed following the standard protocol.

DNA concentration:

• right ITR: 44.5 ng/µL
• left ITR: 32.8 ng/µL

Digestion of ITR fragments with NotI and PstI

Digestion of both fragments was performed as follows:

Incubation: 1 hours + 15 minutes, 37°C

Gel run:

• due to the small fragment sizes, we prepared a 2% agarose gel: 1.3 g agarose + 65 ml TAE buffer
• 25 µL of each sample + 5 µL loading dye (6x); 8µL marker
  
• Expected fragment sizes: ~ 150 bp (both)
• Big problem: Small fragments and little amounts -> 150 bp fragments were nearly not visible. In addition to that, the right ITR sample delivered 2 fragments in the referring range. Therefore two gel extractions ("right ITR oben" and "right ITR unten") were performed.

Gel extraction:

• Sample: right ITR oben = 0.11 g
  
• Sample: right ITR unten = 0.12 g
  
• Sample: left ITR = 0.2 g
  

Gel extraction was performed with special columns provided from Sven. In order to elute the samples 12 µL EB were used, columns were placed for 2 minutes into 50°C water bath and then centrifuged at 13200 rpm for 1 minute.

DNA concentration:

• right ITR oben: 23.9 ng/µL
• right ITR unten: 13.8 ng/µL
• left ITR: 4.46 ng/µL

Hybridization of ITR-Präfix-Oligos

left ITR:

• 10 µL Oligo 1 (O43 = Präfix_for)
• 10 µL Oligo 2 (O44 = Präfix_rev)
• 4 µL 100 mM TrisHCl pH8
• 8 µL 5 mM MgCl2
• 8 µL H2O

Total: 40 mL

right ITR:

• 10 µL Oligo 1 (O47 = Präfix_for)
• 10 µL Oligo 2 (O48 = Präfix_rev)
• 4 µL 100 mM TrisHCl pH8
• 8 µL 5 mM MgCl2
• 8 µL H2O

Total: 40 mL

Because some of the oligos produce strong secondary structures, we used the ORIGAMI1 program:
1) initial denaturation: 99°C, 7 minutes
2) 99°C, 1 minute
3) 73 x repetition of 2) -> -1°C, R = 0.3 °C/sec
4) hold 4°C

Ligation of pGA14 + ITR + Präfix

1. right ITR oben:

• Präfix: 1 µL
• ITR: 7 µL
• vector (pGA14): 9 µL
• buffer: 2 µL
• T4 ligase: 1 µL

Total volume: 20 µL

2. right ITR unten:

• Präfix: 1 µL
• ITR: 7 µL
• vector (pGA14): 9 µL
• buffer: 2 µL
• T4 ligase: 1 µL

Total volume: 20 µL

3. left ITR:

• Präfix: 1 µL
• ITR: 7 µL
• vector (pGA14): 9 µL
• buffer: 2 µL
• T4 ligase: 1 µL

Total volume: 20 µL

Samples will be incubating over night at 16°C.

Ordering genes for the replacement of Rep and Cap parts

Investigator: Volker guided by Sven & Tobi

The replacement for the Rep ORF from base 1510 to base 2008 and for the Cap ORF from base 3064 to base 3985 was ordered from Mr. Gene.

File:Freiburg10 Rep Cap Replacement.pdf

Sequencing of GOI

Investigator: Hanna
This time the sequencing delivered good results:

Conclusion: The designed primers can always be used in order to sequence any gene of interest!!!

57.Labortag 13.07.2010:

Endotoxin-free Midi Prep of pAAV_RC

Investigators: Chris, Stefan, Patrick
Deviations from the Standard Protocol:
As the lysate was applied to the column filter, about 1/5 of the lysate was spilled.
Result of the Midi-prep: about 18 ng/µl => waste
A new Midi-Pred will be prepared for tomorrow: 200 ml LB, 0,2 ml Amp, E.coli XL-1-blue (with pAAV_RC plasmid).

HEK-293 cells

The cells were split according to the standard protocol. Investigator: Patrick

Oligos pMGMK_TK30 arrived

New Oligos arrived for inserting RFC25 to thymidinkinase_gmk construct!(number O60 to O65). They were resuspended in water and a 1:10 dilution was done. They were put in Primer Box 2.

to do: perform PCR tomorrow and clone PCR product into pAAV_RFC25

Transduction

Investigators: Adrian, Patrick

• 3 Plates got transduced

1. Plate I(A is up) completely 10 µg

2. Plate II(A is up)completely 18 µg

3. Plate III(A is up) completely 24 µg

Results of Trafo of cloning CFP into pSB1C3

Investigator: Bea
Results: no clones on trafo plate even after longer incubation at 37 °C
Solution: perform digestion of linearized plasmid pSB1C3 and pMA_CFP again!!!

Repetition of cloning of CFP into pSB1C3

Investigator: Bea

• experiment date: 13.07.2010 ; time: 16:00
  • Vector: name: pSB1c3 number: do not have any number production date: - origin: iGEM HQ sent per DNA distribution kit
  • Insert: name: pMA_CFP number:- production date: - origin: iGEM 2009
• new vector name: pSB1c3_CFP (P48)
• buffer used: 3 ; Restriction-enzymes used: Enzyme 1 (no. Lab: 23) EcoRI ; Enzyme 2 (no.Lab: 48) PstI ;Enzyme 3 (no.Lab: 77) DpnI
• DNA concentration (vector: pSB1C3): 25 ng/µl ; DNA concentration (insert pMA_CFP): 436,8 µg/µl

Comments:The standard plasmid vector (pSB1C3) received from the iGEM HQ is linearized (PCR product) and need to be cut with EcoRI, PstI and optionally with DpnI to digest the DNA template.

Digestion of pSB1C3 and pMA_CFP

• Load pMA_CFP digestion reaction onto 1% agarose gel

1% Agarose-Gel

0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED (3-6µl), at 110 Volt, running time:45 minutes

Loading plan for agarose gel:
Marker used: GeneRuler ladder mix (Fermentas)

Results: Expected bands too high, but lower band (= CFP) was cut out and gel extraction have been performed folowing standard protocol.

Gel extraction

• c(CFP)= 6,75ng/µL

• Ligation ratio of vector/insert was calculated with LabTools
• v(pSB1C3)= 4,4 µL
• v(CFP)= 4,6 µL
• vQuickligase Buffer (2x)= 10µL
• vQuickLigase = 1µL

Transformation

• used bacterial strain: XL-1B
• 2µL of DNA (pSB1C3_CFP)
• Trafo plate in 37°C room for incubating at 23:00

TRANSFORMATION of Quickchange PstI

Investigator: Kira

• Transfection was performed according to the standard protocol with XL1B cells.

58.Labortag 14.07.2010

Trafo evaluation of Quickchange PstI

Investigator: Kira
Both plates contained lots of colonies, while 8-10 colonies are considered for inoculation to perform a mini prep and an additional test digestion. However, the experiment has to be repeated.

Quickchange for the PstI restriction sites in pAAV-RC (modified)

Investigators: Kira

PCR program: PstI

• PCR products were digested with 0.5 µl and incubated @ 37 C for 1 h

TRANSFORMATION

• The transformation of XL1B was performed according to the standard protocol but with DYT instead of LB.

Results of trafo cloning pSB1C3_CFP

Investigator: Bea
Comments: experiment have been repeated because no clones were grown after the last trafo!

• There have been clones grown on the trafo plate. Four clones were picked and inoculated on LB_Medium containing Cm (Chloramphenicol).
• 18:00: 37°C room on shaker. Incubating over night!

Seed AAV-293

Investigators: Adrian ,Bea

• 2x75cm2 culture flasks
• after counting via neubauer chamber (2,2*20*10000*(x/4))= should be around 3,xxx so it would be possible to prepare 4x10cm petri dishes with 3.000.000 cells per plate and passage 1.500.000 cells to a 75cm.

DAAD Timetable

Investigators: Adrian, Kerstin Prepared timetable for DAAD

Summary

Investigators: all

have not been done yet! to do for tomorrow! deadline of submitting project descrpition is 16th of july!! (Bea)

Ordering ITR+TK/GMK

Investigators: Bea, Sven

• It has been ordered the TK30 with NgoMIV add-on prefix and ageI and SpeI add-on suffix combined with the wt_left_ITR in the RFC10 standard.

Endotoxoin free Midi prep mAAV_RC

Investigator: Chris W.
Midi prep done with the Qiagen-Kit
OD of the Bacteria: 3.07
concentration: 429,8 and 378,5 ng/µl
Labeled as P50 and stored in the -20° Freezer

MGMK_TK30:PCR with iGEM restriction-sites and cloning into pAAV_RFC25

Investigators: Achim,Bea,Anissa

PCR:

• PCR program:mGMK_TK30

Comments:Oligos with add-on tails (prefix and suffix), but TK30 still contains three iGEM restriction sites (TK30 without restriction sites was ordered today)

Practical Cloning:

• • Vector: name:pAAV_RFC25 number:p42
  • Insert: name: MGMK_TK30 (after PCR with iGEM standard) number:-
• new vector name: pAAV_RFC25_MGMK_TK30
• buffer used:4 ; Restriction-enzymes used: Enzyme 1 (no. Lab:149) AgeI ; Enzyme 2 (no.Lab:63) XbaI
• DNA concentration (vector):321,5 ng/µl ; DNA concentration (insert):

Comments:_____

Digestion

• Incubation: Vector: 1h; PCR product: 2h

Agarose-Gel:

0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED (3-6µl), at 110 (PCR); 115 (vector) Volt, running time:____ minutes

• Marker: GeneRuler ladder mix

• Marker: GeneRuler ladder mix

Comments: two gels were made, one for the vector, one for the pcr product

• Gel extraction was carried out according to standard protocol
• Nanodrop:
  • pAAV_rfc 25 Digestion: 15,07 ng/µl
  • mgmk tk30 pcr product: 23,04 ng/µl

• Puricifation using Quiagen QIAquick PCR Purification Kit Protocol
  • c(mgmk_tk30) = 8,1ng/µL
• Ligation of mgmk tk30 and pAAV_rfc 25
  • v(vector) = 2,97 µL
  • v(insert) = 6,03 µL

• Trafo
• put in 37°C room at 22:00

Ordering of Oligos

Investigator:Volker

The following four Oligos were ordered:

• SDM BamHI (729) for
• SDM BamHI (729) rev
• SDM SalI (1110) for
• SDM SalI (1110) rev

59.Labortag 15.07.2010

Picking clones for mini-prep

Investigators: Adrian, Chris, Stefan

4 clones (1-4) of pAAV_RFC25_mgmk_tk30 where picked according to protocol for mini-prep (tomorrow).
Antibiotic: Amp
Time: 18:30
Investigators: Stefan, Patrick
New millipore H2O, LB + Agar and DMEM medium were prepared

Splitting of HT1080 cells

Investigators: Chris, Patrick
HT 1080 cells were split according to the standard protocol

PstI Quickchange Trafo from 14.07

Investigator: Kira
Trafo evaluation: Unfortunately no colonies are present.

MINI PREP (PstI Quickchange Trafo from 13.07)

Investigator: Kira

@Kira: ich habe die Clone benannt: SDM310 Nr. 1 ist jetzt P58 und sdm4073 nr 1 ist jetzt P59. Bitte eintragen.

TEST DIGESTION (PstI Quickchange Trafo from 13.07)

Investigator: Kira

FACS

Investigators: Kerstin, Anna

FACS analysis of different treated HT1080 cells was done (18 samples)

The first percentage refers to the living and fluorescent cells and the second percentage to the total amout of living cells.

The data are listed this way:

1. Plate I(A is up)

1= Plate 1 A1 : 0% YFP-positive Cells (there were only a few cells in this sample)
2= Plate 1 A2 : 27,7% YFP-positve Cells from 73,3%
3= Plate 1 A3 : 17,3% " from 63,1%
4= Plate 2 A1 : 32,9% " from 75,3%
5= Plate 2 A2 : 27,2% " from 76,5%
6= Plate 2 A3 : 30,7% " from 73,4%
7= Plate 3 A1 : 26,8% " from 76,0%
8= Plate 3 A2 : 30,5% " from 74,2%
9= Plate 3 A3 : 30,7% " from 75,2%
10= Plate 1 B1 : 6,25% " from 36,1%
11= Plate 1 B2 : 6,42% " from 68,4%
12= Plate 1 B3 : 0,05% " from 89,5%
13= Plate 2 B1 : 20,5% " from 72,5%
14= Plate 2 B2 : 15,7% " from 73,6%
15= Plate 2 B3 : 0,02% " from 85,4%
16= Plate 3 B1 : 19,5% " from 73,3%
17= Plate 3 B2 : 20,8% " from 75,7%
18= Plate 3 B3 : 0,07% " from 84,8%

The calculated value written into the table describes the fluorescent amount of cells from the living cells. Example: 1 A2 : 27,7% YFP-positve cells of 73,3% living cells : 27,7/73,3 = 0,378

1. Plate I(A is up) completely 10 µg

2. Plate II(A is up)completely 18 µg

3. Plate III(A is up) completely 24 µg

Conclusions

• Higher amounts of Plasmids (24, 30µg) seems to have no obvious effect on YFP expression
• Theres no difference in YFP expression comparing resuspended samples to not resuspended samples
• The amount of dead HT cells is about 25%, it is not clear if it is either AAV induced or mechanic stress (Trypsin, washing procedure)

Next steps:

• does the amount of GOI (ng plasmids) has an effect on YFP expression
• compare transduced to non transduced cells (vitality), or in other words is the mechanic stress responsible for 25% dead cell ratio?
• comparison of transduction efficiency of different virus stock production routines

Plasmid Mini-Prep of pSB1C3_CFP

Investigators: Kerstin, Anissa

• experiment date: 15.07.2010 ; time:10.00
• new vector name:pSB1C3_CFP

Glycerol Stocks

Test digestion

• buffer used: 3 ; Restriction-enzymes used: Enzyme 1 (no. Lab:48)PstI ; Enzyme 2 (no.Lab:23) EcoRI
• Plasmid
  • Given Plasmid-Number: p48; DNA concentration: 195,1 ng/µl ;
  • Given Plasmid-Number: p51; DNA concentration: 230,1 ng/µl;
  • Given Plasmid-Number: p52; DNA concentration: 134,1 ng/µl;
  • Given Plasmid-Number: p53; DNA concentration: 244,1 ng/µl;

Comments:p48 and p51 were sequenced

• Incubation: 1h

Agarose-Gel:

0,5 g Agarose, 50 TAE (1%), 3GELRED (3-6µl), at 110Volt, running time:30 minutes

• Marker: GeneRuler ladder mix

Comments: Gel was leaky, therefore two samples "flew through". Plasmid p48 and p51 were sent for sequencing.

2nd try: First steps in producing ITR BioBricks via Fancy Method

Investigator: Hanna

Unfortunately it turned out, that the digestion step of the 1st try didn't work: After the transformation very much colonies grew on the plates. Sequencing delivered, that survival of the bacteria was due to re-ligated vector... Therefore the whole experiment will be performed again today.

Digestion of pGA14_Cerulean-CFP

1. Digestion with EcoRI and NotI:

Incubation: 1.5 hours, 37°C in thermocycler

Gel run: 20 µL sample + 4 µL loading dye (6x); 8µL marker
Expected fragment size: ~ 2900 bp

Gel extraction:
Sample weight: 0.31 g
Gel extraction was performed following the standard protocol. Abberation: After applying EB-buffer, the column was incubated for two minutes at 50°C.

DNA concentration: 43 ng/µL

1. Digestion with XbaI and PstI:

Incubation: 1.5 hours, 37°C in thermocycler

Gel run: 20 µL sample + 4 µL loading dye (6x); 8µL marker
Expected fragment size: ~ 2900 bp

Gel extraction:
Sample weight: 0.31 g
Gel extraction was performed following the standard protocol. Abberation: After applying EB-buffer, the column was incubated for two minutes at 50°C.

DNA concentration: 38.6 ng/µL

Digestion of pAAV_MCS with AlwNI

Incubation: 1.5 hours , 37°C in thermocycler

Gel run: 30 µL sample + 6 µL loading dye (6x); 8µL marker
Expected fragment sizes: 2982 bp = right ITR and 1674 bp = left ITR

Gel extraction:

• Sample: right ITR = 0.19 g
  
• Sample: left ITR = 0.17 g
  

Gel extraction was performed following the standard protocol. Abberation: After applying EB-buffer, the column was incubated for two minutes at 50°C. Further on 30 µL EB-buffer was used.

DNA concentration:

• right ITR: 59.8 ng/µL
• left ITR: 46.2 ng/µL  :)

Digestion of ITR fragments with NotI and PstI

Digestion of right ITR fragment:

Incubation: 50 minutes, 37°C in thermocycler

Digestion of left ITR fragment:

Incubation: 50 minutes, 37°C in thermocycler
Gel run:

• due to the small fragment sizes, a 2% agarose gel was prepared.
• 5 µL loading dye was added to the whole volume of each sample; 8µL marker
  
• 115 V, 40 minutes
  
• Expected fragment sizes: ~ 150 bp (both)
• Problem: Small fragments, little amounts, blurred 150 bp bands -> fragments were cut out broad-minded.

Gel extraction:

• Sample: right ITR oben = 0.4 g
  
• Sample: left ITR = 0.36 g
  

Gel extraction was performed following standard protocol. After applying EB buffer to the columns, they were placed for 2 minutes into 50°C water bath and then centrifuged at 13200 rpm for 1 minute.

DNA concentration:

• right ITR: 4 ng/µL
• left ITR: 4 ng/µL

Hybridization of ITR-Präfix-Oligos

left ITR:

• 10 µL Oligo 1 (O43 = Präfix_for)
• 10 µL Oligo 2 (O44 = Präfix_rev)
• 4 µL 100 mM TrisHCl pH8
• 8 µL 5 mM MgCl2
• 8 µL H2O

Total: 40 mL

right ITR:

• 10 µL Oligo 1 (O47 = Präfix_for)
• 10 µL Oligo 2 (O48 = Präfix_rev)
• 4 µL 100 mM TrisHCl pH8
• 8 µL 5 mM MgCl2
• 8 µL H2O

Total: 40 mL

Because some of the oligos produce strong secondary structures, we used the ORIGAMI1 program:
1) initial denaturation: 99°C, 7 minutes
2) 99°C, 1 minute
3) 73 x repetition of 2) -> -1°C, R = 0.3 °C/sec
4) hold 4°C

Ligation of pGA14 + ITR + Präfix

1. right ITR

• Präfix: 2 µL
• ITR: 10 µL
• vector (pGA14): 5 µL
• buffer: 2 µL
• T4 ligase: 1 µL

Total volume: 20 µL

2. left ITR

• Präfix: 2 µL
• ITR: 10 µL
• vector (pGA14): 5 µL
• buffer: 2 µL
• T4 ligase: 1 µL

Total volume: 20 µL

3. control 1:

• vector 1 (pGA14, digested with EcoRI + NotI): 5 µL
• buffer: 2 µL
• T4 ligase: 1 µL
• H2O: 12 µL

Total volume: 20 µL

4. control 2:

• vector 2 (pGA14, digested with XbaI + PstI): 5 µL
• buffer: 2 µL
• T4 ligase: 1 µL
• H2O: 12 µL

Total volume: 20 µL

Samples will be incubating over night at 16°C. To do: Trafo (Agar plates were already prepared.)

60.Labortag 16.07.2010

Continuation of Fancy ITR-method

Investigator: Hanna

Trafo

Trafo was performed following standard protocol. Aberration: Instead of 2 µL, 3 µL DNA was added to BL21 bacteria (50 µL).

right ITR

It turned out, that the strategy by digesting pGA14 with XbaI and PstI cannot work. pGA14 needs to be digested with EcoRI and PstI:

Digestion of pGA14_Cerulean-CFP with EcoRI and PstI

Incubation: 1 hour, 37°C in thermocycler

Gel run: 1% gel, 20 µL sample + 4 µL loading dye (6x); 8µL marker
Expected fragment size: ~ 2900 bp

Gel extraction:
Sample weight: 170 mg
Gel extraction was performed following the standard protocol. Abberation: After applying EB-buffer, the column was incubated for two minutes at 50°C.

DNA concentration: 26.8 ng/µL

Digestion of pAAV_MCS with NotI and PstI

In order to create ITRs with NotI and PstI restriction sites, the AlwNI-digestion step was skipped. Therefore 50% of the constructs will also contain the left ITR... this means: picking more colonies and sequencing!

Incubation: 1 hour, 37°C in thermocycler

Gel run: 2% gel, 20 µL sample + 4 µL loading dye (6x); 8µL marker
Expected fragment size: ~ 150 bp
Gel run delivered no satisfying results. Therefore the gel was discarded and the strategy changed: The residual right ITR solution of yesterday's elution will be taken for ligation.

Ligation of right ITR + Präfix + pGA14

• ITR: 7 µL
• pGA14: 7 µL
• Präfix: 1.5 µL
• Buffer: 2 µL
• T4 ligase: 1 µL
• H2O: 1.5 µL

Total: 20 µL

• pGA14: 7 µL
• Buffer: 2 µL
• T4 ligase: 1 µL
• H2O: 10 µL

Total: 20 µL

The ligation-solution will be incubating over night.

To do:

• Mini-Prep of pGA14_präfix_leftITR
• Trafo of pGA14_präfix_rightITR
• Midi-Prep of pGA14

Results sequencing of pSB1C3_CFP

Investigator: Bea
Plasmid: pSB1C3_CFP
Primer used: VR2
Plasmid names: P48_KK and P51_AB
Comments: Sequencing results of cloning experiment performed at 14.-15.07.2010.

Results: Sequencing reads are good. Cloning of CFP into pSB1C3 can be confirmed. There are no insertions, transitions or deletions in the sequence or the iGEM restriction sites. Using this plasmid in order to create our iGEM BioBricks in RFC10 standard can be confirmed.

Sequencing results of pSB1C3_CFP (plasmid number P48). There are no mutations in the CFP sequence and in the RFC10 iGEM restiction sites (blue boxes).

Sequencing results of pSB1C3_CFP (plasmid number P51). There are no mutations in the CFP sequence and in the RFC10 iGEM restiction sites (blue boxes).

BioBrick production with hGH and beta-globin by cloning it into pSB1C3

Investigator: Bea
Plasmid: pSB1C3_CFP
Plasmid names: pSB1C3 (P51 or P48) and PCR prdouct of hGH and beta-globin

Comments: While cloning theoretically the hGH PCR product into the vector pSB1C3, it was recognized that the primers that were used for PCR for

• hGH RFC10 production and
• beta-globin intron RFC10 production

do not contain the required "G" at the end of the prefix and the "T" in the suffix add-on in the RFC10 standard. They were already used by performing a PCR with the add-on prefix and suffix primer, but we did not clone it into the pSB1C3 because of the wrong add-on tails. Therefore new primers were designed and ordered.

Ordered oligos:

hGH

• suffix (reverse)
• prefix (forward)

beta-globin

• suffix (reverse)
• prefix (forward)

CMV

• suffix (reverse)
• prefix (forward)

TK_GMK

• tk_gmk_prefix for
• tk_gmk_suffix_rev
• gmk_suffix_rev

To do: Upload cloning strategy of tk_gmk and BioBrick production

--Bea 15:21, 16 July 2010 (UTC)
Comment: reverse beta-globin primer is correct!? (Hanna)

Transfection

Investigator: Adrian
30µg Plasmid were used (pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30)
VIRUS nix:pHelper + pAAV_RC
VIRUS 1  :pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30 clone 1 : P54
VIRUS 2  :pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30 clone 2 : P55
VIRUS 3  :pHelper + pAAV_RC+ pAAV_RFC25_mgmk_TK30 clone 3 : P56

Mini Prep of pAAV_RFC25_mgmk_TK30

Investigator: Adrian 4ml were preparated

• pAAV_RFC25_mgmk_TK30 clone1 conc: 291,43 ng/µl : P54
• pAAV_RFC25_mgmk_TK30 clone2 conc: 243,99 ng/µl : P55
• pAAV_RFC25_mgmk_TK30 clone3 conc: 247,00 ng/µl : P56
• pAAV_RFC25_mgmk_TK30 clone4 conc: 246,01 ng/µl : P57

Test digestion of all four plasmids to check if the mgmg_TK30 insert is present. Investigator: Patrick, Chris
Each digestion sample:

• 0,2 µl BSA (I picked the wrong tube. It should have been 2 µl BSA)
• 2 µl Buffer 4
• 0,5 µl XbaI
• 0,5 µl SpeI
• 13,8 µl H2O
• 3 µl DNA

Digestion: 65 minutes, 37°C. Results: No mgmk_TK30 insert detectable. I will try it again on saturday or sunday.

Preparation of 50x TAE and 0,5M EDTA

Investigators: Chris W., Patrick

1 L TAE (50x)

242 g tris base
57.1 ml glacial acetic acid
100 ml 0.5 M EDTA
ad 1 L Milipor

1 L EDTA (0.5 M)

146,13 g Ethylendiamintetraessigsäure
ad 800 ml Milipor
~20 g NaOH
pH mit NaOH auf 8 einstellen
ad 1 L Milipor

Test digestion of the QuickChange for PstI (310) and PstI (4073)

Investigator: Kira & Volker

The digestion showed two bands with apropirate lengt for each of the 6 clones as they were expected.
The constructs were sent to GATC for sequencing.

• pAAV_RC_SDM_PstI(310) with the primer GATC_std_QE-FP
• pAAV_RC_SDM_PstI (4073) with the primer GATC_std_M13_FP

Primers to convert the expression constructs into the iGEM standard were ordered

Investigator: Volker

• Praefix VP1ex
• Praefix VP2ex
• Praefix VP3ex
• Suffix VP123ex
• Praefix Rep68_78ex
• Praefix Rep 40_52ex
• Praefix AAPex
• Suffix Rep 40_68ex
• Suffix Rep 52_78ex
• Suffix AAPex

61.Labortag 17.07.2010:

Picking clones of pGA14_Präfix-leftITR Trafo

Investigator: Hanna

Bacteria also grew on control plates. Control means: Ligation of digested vector (pGA14_Cerulean) without insert (Präfix and left ITR). Therefore it seemed that somehow pGA14 religated (Can't be if vector digested completely). Therefore 10 colonies were picked, in order to increase the chance to get a successfully ligated plasmid.

Mini-Preps of pGA14_Präfix-lITR cultures

Investigator: Hanna

No glycerol stocks were prepared, because I'm going to perform the test digestion today.

10 Mini-Preps were performed following the standard protocol.

Test digestion of pGA14_Präfix-lITR (Ligation)

Investigator: Hanna

Test digestion

• buffer used: 4; Restriction-enzymes used: Enzyme 1: EcoRI-HF ; Enzyme 2: PstI-HF
• Plasmid: pGA14_Präfix_lITR 1 - 10 (all: ~ 400 ng/µL)

• Incubation: 55 minutes

Agarose-Gel:

0.75 g Agarose, 50 ml TAE (1.5 %), 3 µL GELRED, at 115 Volt, running time: 30 minutes

Expected fragment size: Hopefully 160 bp = left ITR :) If pGA14_Cerulean religated the expected fragment size would be ~ 800 bp.

• Marker: GeneRuler ladder mix (6x)

Comments: Numbers refer to "colony 1, 2, 3,..."

Wonderful news: All 10 test digestions delivered positive results: 150 bp fragments and no 800 bp fragment was detectable. Means: left ITR + Präfix was inserted successfully into pGA14!!! :)

yees, thats great! Finally something worked with our ITRs. well done Hanna!! (Bea)

the most astonishingly test digestion gel of our entire working time! :)(Adrian)

Next step: Fusion of suffix to left ITR

Glycerol stocks of all 10 colonies were prepared: B46.1 - B46.10; Plasmid names: P62.1 - P62.10

Trafo of pGA14_Präfix_rITR

Investigator: Hanna

Transformation of BL21 bacteria with pGA14_Präfix_rITR which ligated over night was performed following standard protocol. Abberation: 3µL DNA was added to 50 µL cells.

To do: Mini-Prep + Test digestion of pGA14_Präfix_rITR; Sequencing of pGA14_Präfix_lITR and pGA14_Präfix_rITR.

62.Labortag 18.07.2010

Investigator: Adrian

Calculation of ganciclovir cymeven concentration

• Ganciclovir: m = 255,23 g/mol
• dissolved in PBS (?), NaCL (0,9%) ?
• Concentrations : 0,5 µM = 0,0000005M
• xg = (0,0000005 mol/l *255,23 g/mol)/(1l PBS resp. 1l 0,9% NaCl)
• x = 0,000128g
• x = 0,128 mg
• 0,128 mg solved in 1l of PBS = 1l of 0,5µM solution
• steril filtrated under steril bench

preparation of Aliquots:

Cellculture

The HEK-293 cells were split according to the standard protocol. The HT 1080 cells were split and sowed (3x10^5 cells each well). The amount of cells/ml was determined with the Neubauer-Meteringchamber: (8 cells counted/4)x2,2x20x10^4 = 888000 cells/ml. Two 6-well dishes could be prepared.

Test digestion of pAAV_RFC_mgmk_tk30: P54, P55, P56, P57

Investigators: Chris, Patrick

To check whether the mutual mgmk_tk30 insert is present in the plasmid we digested it with AgeI and EcoRI. If the insert is present there will an DNA fragmet with a size of about 1700 bp.

Mastermix:

• 10 µl Buffer 4
• 4,4 µl AgeI
• 2,5 µl EcoRI
• no BSA (accroding to NEB)
• 83,1 µl H2O
• Total volume: 100 µl

17 µl of the mastermix were pipetted to 3 µl plasmid sample. Digestion time: 75 minutes.

Gelrun (1% Agarose), 55 minutes, 115 mV: Results: See picture. Interpretation of the results: ? The "upper" DNA seems to be undigested vector underneath the probably digested vector with an estimated size of about 5600 bp. The mutual insert (mgmk_tk30) should be about 1700 bp. There are probably several DNA coil structures visible but not our mutual insert. To do : ligate again.

Aferwards i noticed that i miscalculated the Mastermix.

63. Labortag 19.07.2010

rITR

Picking clones of pGA14_Präfix-rITR Trafo

Few bacteria also grew on control plates. Control means: Ligation of digested vector (pGA14_Cerulean) without insert (Präfix and right ITR).
6 colonies were picked, in order to increase the chance to get a successfully ligated plasmid.

Mini-Preps of pGA14_Präfix-lITR cultures

Investigators: Hanna, Jessy

Glycerol stocks were prepared: B47.1, B47.2, B47.3, B47.4, B47.6

6 Mini-Preps were performed following the standard protocol.

Test digestion of pGA14_Präfix-rITR (Ligation)

Investigator: Hanna, Jessy

Test digestion

• buffer used: 4; Restriction-enzymes used: Enzyme 1: EcoRI-HF ; Enzyme 2: PstI-HF
• Plasmid: pGA14_Präfix_rITR 1 - 6 (all: ~ 500 ng/µL) -> 1 µg

• Incubation: 45 minutes

Agarose-Gel:

0.75 g Agarose, 50 ml TAE (1.5 %), 3 µL GELRED, at 90 - 100 Volt, running time: 45 minutes

Expected fragment size: Hopefully 160 bp = right ITR :) If pGA14_Cerulean religated the expected fragment size would be ~ 800 bp.

• Marker: GeneRuler ladder mix (6x)

Comments: Numbers refer to "colony 1, 2, 3,..."

Wonderful news: All 6 test digestions delivered positive results: ~ 160 bp fragments and no 800 bp fragment were detectable. Means: right ITR + Präfix was inserted successfully into pGA14!!! :)

Next step: Ordering new oligos: Präfix of lITR, suffix of right and left ITRs, continuation of fancy cloning protocol

New Oligos (left ITR Präfix and Suffix + right ITR Suffix) were ordered.

Sequencing of ITRs

Investigator: Hanna

pGA14_Präfix_lITR and pGA14_Präfix_rITR were sent for sequencing to GATC:
1) pGA14_Präfix_lITR: c(P62.1) = 422.38 ng/µL -> V = 5 µL + 25 µL H2O
2) pGA14_Präfix_rITR: c(P63.1) = 501 ng/µL -> V = 4.2 µL + 25.8 µL H2O

Primer: GATC_std_pQE-FP

Quantitative real time PCR

Investigator: Hanna

Template: DNA of HT1080 cells transducted with 1) 10 µL and 2) with 30 µL virus stock solution.

Pipetting scheme:
Master Mix

• 2x QuantiFast SYBR Green PCR Mix: 400 µL
• Primer for and rev: 32 µL of 1:10 dilution = 10 µM
• Water: 208 µL
• Total: 640 µL

Template: 5 µL
To each template 20 µL master mix were added.

PCR program:

• Hold: 95°C, 5 minutes
• Cycling: 95°C for 10 seconds, 60°C for 30 seconds
• Melt: Ramp from 60°C to 95°C, rising by 1°C each step, wait for 45 seconds on first step, then wait for 5 seconds for each step afterwards. Aquire to Melt A on FAM.

Cloning strategy for gmk_tk added!!

Investigator: Bea
Media:Freiburg10 BioBrick GMK TK30 07 07 2010 with new oligos.pdf

pAAV_RC site directed mutagenesis: Digestion of the two mutagenesis reaction of PstI site: 310 and 4073

Investigator: Volker

Digestion of pAAV_RC

Incubate at 37°C

• Gelextraction was prepared according to the protocol
  
  • weight of gel: 310: 150 mg; 4073: 150mg
    
• Nanodrop: 310: 5,5ng/µl; 4073: 7,0ng/µl
  
• Ligation was prepared according to the protocol
  
  • Ratio: 310:4073 --> 2,52 µl:6,48µl
    

Ligation was frozen in -20°C

pHelper plasmid for Midi-Prep prepared

Investigator: Stefan
200ml DYT media + 200µl Amp were inoculated with pHelper-plasmid glycerol stock and put on shaker in 37°C room.
For Midi-Prep: Use Endotoxin-free Midi Prep not our Midi Prep!

Trafo of IGEM 2009 linkers

• We borrowed several linkers/tags from Gerrit and did a Trafo in XL1B cells:
  • small linker
  • middle linker
  • long linker
  • Strep-Tag
  • Split
  • GSAT
  • SEG

• Amount of DNA used: 0,5 µl
• Plates incubated at 37°C overnight

64. Labortag 20.07.2010

Quickchange site directed mutagenesis:

Investigators: Achim,Anissa
1) VP1ex P27.2
2) VP2ex P28.2
3) VP3ex P29.2
PCR reaction:

• 2.5 µL 10x Pfu Ultra II buffer
• 1) 1,44µl 2) 1,83µl 3) 1,76µl template (1:100 dilution)
• 0.34 µL primer SDM PstI (4073)forward (of 1:10 dilution)
• 0.35 µL primer SDM PstI (4073) reverse (of 1:10 dilution)
• 0,5 µL DMSO (primers form very strong secondary structures)
• 0.5 µL dNTP
• 1) 18,9 2) 18,5 3) 18,6 µL dH2O
• 0.5 µL PfuUltra II fusion (1.25 U)

-> end volume: 25 µL

PCR program:
Quickge
Trafo was carried out, using 2µl DNA, strain:XL1 blue

Transformation with ligation from july 19th

• used bacterial strain: BL21
• 2µL of DNA (pAAV_RC w/o PstI 310/4073)
• Trafo plate in 37°C room for incubating at 12.30
• no clones to pick at 22.00

Picking Clones from Linker Trafo

• clones were picked at 22.00

Endotoxin-free Midi Prep of pHelper

Investigators: Chris, Stefan
Midi-Prep was done according to protocol.
Result of the Midi-prep: 503,2 ng/µl
Plasmid (P64) was placed in Stratagene-Box.

Quantitative real time PCR

Investigator: Hanna

Because there was one positive non-template control (H2O) yesterday, we performed the qPCR one more time.
For this purpose we also calculated the plasmid pipetting for the standard curve again:

• c(P9) = 272 ng/µL --> 3 x 1:10 = 1:1000 dilution.
• 2.68 µL of the 1:1000 dilution was added to 52.32 µL H2O = 2600000 copies/µL.
• By performing 1:10 dilutions further 6 "standard samples" were created: 260000 copies/µL; 26000 copies/µL; 2600 copies/µL; 260 copies/µL, 26 copies/µL, 2.6 copies/µL

Template: DNA of HT1080 cells transducted with 1) 10 µL and 2) with 30 µL virus stock solution.

Pipetting scheme:
Master Mix

• 2x QuantiFast SYBR Green PCR Mix: 400 µL
• Primer for and rev: 32 µL of 1:10 dilution = 10 µM
• Water: 208 µL
• Total: 640 µL

Template: 5 µL
To each template 20 µL master mix were added.
Comment: To non-template control 2 17 µL + 3µL H2O was added (not enough master mix was left).
PCR program:

• Hold: 95°C, 5 minutes
• Cycling: 95°C for 10 seconds, 60°C for 30 seconds
• Melt: Ramp from 60°C to 95°C, rising by 1°C each step, wait for 45 seconds on first step, then wait for 5 seconds for each step afterwards. Aquire to Melt A on FAM.

Cloning of p51(pSB1C3_RFC25) for His-tag insert

Investigators: Anna, Stefan

1% Agarose-gel, 3µl Gelred

Loading dye(5x): 4µl

Loading plan: marker (10µl), free, sample (24µl)

expected size of fragment: 2080 bp

65. Labortag 21.07.2010

Sequencing of ITRs

Investigator: Hanna

P62.1 (=left ITR + RFC23-Präfix in pGA14) and P63.1 (= right ITR-Präfix in pGA14) were sequenced:

Sequencing results of pGA14_lITR-RFC23Präfix.

Sequencing results of pGA14_rITR-RFC10Präfix).

Sequencing delivered, that the präfixes were fused successfully to the ITRs. The mismatch (orange box) was due to the alignment with the RFC10 präfix... (oligos were ordered!), what means, that the sequencing is OK.
Interestingly it seems, that not only we had problems with PCR of the ITR -> Very strong secondary structures. Even sequencing of the ITRs seems impossible under normal conditions: Exactly at the point where the ITR sequence starts the sequencing signal delivered no clear results.

Conclusion: Fancy method worked until now.

Quantitative real time PCR

Investigator: Hanna

Because the non-template control and the not-transduced cell-DNA controls delivered positive PCR results for the CMV-promoter, a control qPCR was prepared:
1. not-transduced cell-DNA controls (K1 and K2) were diluted: 1:100
2. Oligos (O20, O21) were 1:10 diluted
3. Master Mix was prepared:

• 75 µL SYBR Green
• 39 µL H2O
• 6 µL Oligos

4. Pipetting scheme:

• 1. K1
• 2. K2
• 3. NTC1
• 4. NTC2
• 5. Sdt7

Plasmid Mini-Prep

• experiment date: 21.07.2010 ; time: 10:30Uhr
• name of investigator: Anissa, Kerstin

Glycerol Stocks

Comments: Different Linker-fragments are very short (5-178bp) --> no Test-digestion; directly sequencing

Results of Trafo of cloning pAAV_RC SDM of PstI 310 and 4073

Investigator: Bea
Comments: Cloning together of the two site-directed mutagenesis performed with the pAAV_RC

Results: no clones grown on LB-agar plate, because pAAV_RC was digested with only one blunt-end restriction enzyme. Luckily, th enzyme vut twice, and one time in the Amp gene. Therefore wrong ligation of the two fragments lacked the Amp resistance.

To do: new cloning together of pAAV_RC PstI 310 and 4073 with TWO other enzymes.

PCR with pUB_V5_mGMK_TK30

Investigator: Adrian, Bea
Comments: Perform PCR with two primers

• pmGMK_TK30_prefix_RFC25_for
• pmGMK_TK30_suffix_RFC25_rev

and pUB_V5_hisB_mGMK_tk30 (P15) and clone it into pAAV_RFC25.

PCR:

• PCR program: GMKTK

Comments:New Oligos (pmGMK_TK30_prefix_RFC25_for and pmGMK_TK30_suffix_RFC25_rev) with add-on tails (prefix and suffix), but TK30 still contains three iGEM restriction sites but will be ready for use in cell culture
Result of PCR product (theoretically)

Digestion of pAAV_RFC25

• Vector: name:pAAV_RFC25 number:p42
• new vector name: pAAV_RFC25_MGMK_TK30
• buffer used:4 ; Restriction-enzymes used: Enzyme 1 (no. Lab:149) AgeI ; Enzyme 2 (no.Lab:63) XbaI
• DNA concentration (vector):321,5 ng/µl

Comments: Digestion of pAAV_RFC25 wihle performing PCR with P42

• Incubation: Vector: 1.5 h

Agarose-Gel:

• 0,5 g Agarose
• 50 ml TAE (1%)
• 3 µl GELRED (3-6µl)
• 110 Volt, running time: 50 minutes

Results of digestion with XbaI and AgeI of pAAV_RFC25 and PCR with pUB_V5_gmk_tk30

Digestion of pAAV_RFC25 witth XbaI and AgeI and PCR of pUB_V5_gmk_tk30 with prefix and suffix primer in RFC25 standard loaded on 1% agarose gel.

Result: Gel was loaded with 50µL of pCR product and 20µL of pAAV_RFC25 digestion. Expected bands can be seen, but pAAV_RFC25 too high and there are some additional bands. We cut out only the intensive band at around 1700 bp.

Comment:After a PCR there can be two ways to proceed with the PCR product:

1. load 7-8µL of PCR sample on an analytical gel and watch agarose gel for additional bands. If there are some more bands than expected load sample on preparative gel.If there aren´t any additional bands. proceed with digestion of PCR product in order to ligate it into vector.
2. load total PCR sample on preparative gel. Ask Sven or Tobi in which volume you should elute after cutting out the gel (Gel extraction), but you should elute sample at least in 30µL.

• After gel extraction was performed, concentration of the two constructs was measured and revealed:
  • c(pAAV_RFC25)= 27,33ng/µL
  • c(gmk_tk30)= 24,53ng/µL
• After the gel extraction, the PCR product gmk_tk30 was digested again with XbaI and AgeI
• and purified with the PCR purification Kit provided by Qiagen

Digestion of the PCR product gmk_tk30

• Incubation: 1:45 h
• Ligation and Trafo were performed following standard protocols.

Gelextraction, Ligation and Transformation for cloning of pSB1C3_His_RFC25 (continuation of cloning from 64th lab day)

Investigator: Bea, Kerstin, Stefan

Extraction

• Gel extraction performed following standard protocol provided by Qiagen

Ligation

• Measure DNA-concentration with Nanodrop
• c(pSB1C3) = 18,2,8 ng/µL
• c(His-tag) = 11,2 ng/µL
• Calculation of volume needed for ligation:
• c(pSB1C3) = 8,26 µL
• c(His-tag) = 0,74 µL

Transformation

• Transformation has been followed the standard protocol Media:Freiburg10_Cloning Protocol.pdf
  Deviation of standard protocol added: medium added to cells before incubation on thermomixer changed to DYT. Standard protocol changed.

Picking clones pf VP1-3 pEX plasmids from DKFZ

Investigator: Bea
Two clones of each plasmid were picked and inoculated in 10mL DYT medium containing 10 µL ampicillin

• VPex-1 clone 1
• VPex-1 clone 2
• VPex-2 clone 1
• VPex-2 clone 2
• VPex-3 clone 1
• VPex-3 clone 2

and they were put in the 37°c room at 19:15

66. Labortag 22.7.2010

cell culture

Investigator: Adrian HT 1080 cells were split according to the standart protocol.

Mini Prep and test digestion pKEX_VP1-3 w/o PstI(4073)

Investigators: Kerstin, Jessica
PstI should be deleted in VP1, VP2, VP3

• Glycerol stock were prepared:
  • B55-B60
• Plasmids
  • pKEX_VP1 clone1:488,2 P72
  • pKEX_VP2 clone1:484,8 P73
  • pKEX_VP3 clone1:485,8 P74
  • pKEX_VP1 clone2:499,2 P75
  • pKEX_VP2 clone2:511,7 P76
  • pKEX_VP3 clone2:470,4 P77
    

Test digestion of three plasmids to check if the PstI 4073 is deleted (compared to the origin P27-P29)

Digestion: 45 minutes, 37°C. Results:

Deletion was succesful, there are still 2 PstI restriction sites in P72-74

Cloning together of pAAV_RC PstI 310 and 4073 with two enzymes

Investigators: Chris, Patrick

Clone P58 (pAAV_RC_SDM310.1) and P59 (pAAV_RC_SDM4073) will be digested with HindII and SpeI. The smaller fragment (about 1700 bp) of P58 and the larger fragment of P59 (about 5900 bp) will be ligated to receive a plasmid carrying both mutations.

Incubation: at 37°C, 70 minutes.

The Gelextraction was performed according to the standard protocol.
Weight of P58 gel extract: 50 µg
Weight of P59 gel extract: 98 µg
The received concentrations (measured by nanodrop): P58: 3 ng/µl , P59: 4,7 ng/µl
Quickligation was not performed according to the standard protocol:

• 2 µl P58 Insert
• 2 µl P59 Vector
• 5 µl Quickligase buffer (2x)
• 1 µl Quickligase
• Total volume: 10 µl

Incubation: 15 minutes at roomtemperature.
Transformation was performed according to the standard protocol with E. coli BL21. The agar plates were put into the 37°C room at 23:00.
New Ampicillin was prepared and put into the -20°C freezer.

Sequencing of linker

Investigator: Anissa

middle linker:

GSAT linker:

long linker:

Split linker:

Strep tag:

Short linker:

SEG linker:

Biobricks of hgH and ß-Globin

Investigators: Stefan, Anna

Could you upload some agarose gels eg the gels after the digestion and the PCR? Thanks a lot! (Bea)

Comments: Perform PCR with two primers

PCR of ß-Globin and hgH:

• DNA template: pAAV_MCS; concentration: 360 ng/µl

• PCR program: BGLOB

• PCR program: HGH

Digestion of pSB1C3_RCF25_CFP:

• DNA-Concentration: 230,9 ng/µl

Gel running of digested vector and PCR products

• 1% Agarose gel
• 3 µl Gelred, 8 µl DNA-Ladder-Mix
• 125 Volt, running time: 40 minutes

Gelextraction

• Gel extraction performed following standard protocol provided by Qiagen

Digestion of PCR products

Purification of PCR products, Ligation, Transformation

procedure has been followed a different protocol (see Sven); has to be added in the standard protocols !

• Measure DNA-concentration with Nanodrop:

c(pSB1C3) = 12,42 ng/µL
c(ß-Globin) = 51,93 ng/µL
c(hgH)= 36,87 ng/µL

• Calculation of volume needed for ligation:

c(pSB1C3) = 7,3 µL
c(ß-Globin) = 1,7 µL

c(pSB1C3) = 7,62 µL
c(hgH) = 1,38

Note: after heatshock cells were put on the shaker without adding 750 µl DYT, it was recognized and added later after 25 min of shaking !

Picking clones of pSB1C3_His_RFC25 and pAAV_RFC25_GMK_TK30 for Mini-Prep

Investigators: Stefan

Three clones of each construct were picked and inoculated in 10mL DYT medium containing 10 µL ampicillin (pAAV_RFC25_GMK_TK30) or 10 µL chloramphenicol (pSB1C3_His_RFC25).

They were put in the 37°C room at 20:33.

67. Labortag 23.7.2010

Picking clones of pAAV_RC_1.1

Investigator: ChrisW, Patrick
The inoculated agar plates from 'Cloning together of pAAV_RC PstI 310 and 4073 with two enzymes' were checked for colonies. Three clones were picked and in each case inoculated 20 ml DYT + Amp at 10:20.

Mini prep of two pAAV_RC_SDM_PstI_310_4073. Results: It was checked visually whether enough E. coli BL21 cells had grown. Therefore only two midi preps were carried out, according to the standard protocol (Final elution with 50 µl). Unfortunately both of them yielded a quite low plasmid concentration, probably due to the short growth time (7 h).
pAAV_RC mini pstI 301,4073: 44,7 ng/µl
pAAV_RC mini 2 pstI 301,4073: 83,1 ng/µl

30 µl of both plasmids were sent for sequencing to GATC, named PS1 and PS2. Primers used: GATC_std_PolyC-D and GATC_std_SK.

Glycerol stock:
B 67: pAAV_RC_SDM_PstI_310_4073 (1)
B 68: pAAV_RC_SDM_PstI_310_4073 (2)

Results of Trafo Biobricks of hgH and ß-Globin

Investigator: Anissa, Bea

Comments: No clones of pSB1C3_hGH and pSB1C3_betaGlobin grown on trafo plate. Possible reason can be the forgotten DYT medium added to the cells after the heat-shock. Cells were incubated at 37°C for 40 minutes without medium and were added afterewards.

TO DO: Repeat ligation of

• hgh PCR product and
• beta-globin PCR product

• Measure DNA-concentration with Nanodrop:

c(pSB1C3) = 38,9 ng/µL
c(ß-Globin) = 51,93 ng/µL
c(hgH)= 36,87 ng/µL

• Calculation of volume needed for ligation:

c(pSB1C3) = 3,8 µL
c(ß-Globin) = 5,2 µL

c(pSB1C3) = 3,8 µL
c(hgH) = 5,2

Attention: There was not enough cut vector. In both Ligations were only 2,2 µl of vector added. So incubation lasted 15 minutes and for transformation 4 µl were used.

Mini-Prep and Test digestion of pAAV_RFC25_mGMK_TK30

Investigator: Adrian, Bea

Comments: Mini-Prep, test digestion and sequencing is carried out in order to verify insertion of the PCR product mGMK_TK30 with RFC25 add-on tails into pAAV_RFC25(overhangs). Aim is to use the construct in order to conduct experiments with the gmk_tk30 fusionenzyme.

Mini-Prep of pAAV_RFC25_mGMK_TK30 clone 1 to 3: After performing Mini-Preps of clone 1 - 3 (elution volume with EB buffer was 50 µL) , concentration was measured and revealed following concentrations:

• P81 = pAAV_RFC25_mGMK_TK30 clone 1: c = 486 ng/µL
• P82 = pAAV_RFC25_mGMK_TK30 clone 2: c = 426 ng/µL
• P83 = pAAV_RFC25_mGMK_TK30 clone 3. c = 457 ng/µL

Test digestion of pAAV_RFC25_mGMK_TK30 clone 1 to 3:

• P81 = pAAV_RFC25_mGMK_TK30 clone 1
• P82 = pAAV_RFC25_mGMK_TK30 clone 2
• P83 = pAAV_RFC25_mGMK_TK30 clone 3

Expected sizes:

• mGMK_TK30: 1700bp
• pAAV_RFC25: 4600bp

Results of samples loaded 1% agarose gel and ran 50 minutes at 110 V:

Sequencing of pAAV_RFC25_mGMK_TK30

70ng/µL were sent to GATC from each clone. They were labeled:

• AF_P81 (clone 1)
• AF_P82 (clone 2)
• AF_P83 (clone 3)

Mini-Prep of pSB1C3_His6

Investigator: Adrian

pSB1C3_6xHis clone 1 = P84 : 107,8 ng/µl (eluted with 50µl)
pSB1C3_6xHis clone 2 = P85 : 122,8 ng/µl (eluted with 50µl)
pSB1C3_6xHis clone 3 = P86 : 98,8 ng/µl (eluted with 50µl)

68. Labortag 24.7.2010

Cell culture: Seeding of HEK293 for Transfektion with P81, P82 and P83

Investigator: Adrian

• unfortunately the T75 flask with HEK-cells was overpopulated => to much cells =>yellow medium and differentiation of the HEK-cells => thrown away
• I had to thaw cells, (P4) so there'll be no transfection at Monday :(

Cell culture: Transduction of HT1080 with AAVs

Investigator: Adrian

I. Plate I(A is up)

II. Plate II(A is up)

III. Plate II(A is up)

Discussion round nr. 4

Investigators: Adrian and his evil twins

• adrian created a poster

Stocktaking of superior goods

Investigator: Adrian
superior goods:

• DYT/LB medium
• tips
• LB-agar

69. Labortag 25.7.2010

Clone picking from pSB1C3_hgH and pSB1C3_betaglobin

Investigator: Bea

Comments: Clones were very small and not many clones could be detected. Possible reasons:
1) The ratio for a proper ligation need to be 3:1 insert: vector. There was not enough plasmid of the digested vector pSB1C3.
2) The digested fragments of the PCR products hGH and beta-globin and the cutted vector were stored at -20°C. This could lead to the bad results of the transformation, because the overhangs were degraded.

Two clones were picked of each trafo plate:

• pSB1C3_hGH
• pSB1C3_betaglobin

They were inoculated in 10mL DYT medium containing 10µL chloramphenicol and put into 37°C room for incubating. The reason for picking only two clones was that not many more clones could be detected easily.

70. Labortag 26.7.2010

Mini Prep and test digestion pSB1C3_hgh and pSB1C3_beta-globin

Investigators: Anissa, Kerstin

• Glycerol stock were prepared:
  • B69-B71
• Plasmids
  • pSB1C3_hgh clone1: 139 ng/µl p89
  • pSB1C3_hgh clone2: 209,4 ng/µl p90
  • pSB1C3_beta-globin clone1: 217,5 ng/µl p91
    

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

Test digestion with Xba and PstI to check if hgh and beta-globin were cloned succesfully into the pSB1C3, respectively.

Test digestion P87: pAAV_RC_PstI-301-4073 clone 1 and P88: pAAV_RC_PstI-301-4073 clone 2

Investigator: Chris W.

• Plasmids
  • pAAV_RC_PstI-301-4073 clone1: 44,2 ng/µl p87
  • pAAV_RC_PstI-301-4073 clone2: 83,2 ng/µl p88
  • pAAV_RC: 429,8 ng/µl p50
    

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

Test digestion with PstI 0,5µl

Oligos for hTERT promoter

Investigator: Bea

Oligos were ordered to modify hTERt promoter in order to obtain hTERT in the iGEM RFC10 standard.

Ordered oligos at Sigma-Aldrich for amplifying hTERT promoter.

Retrafo with pTVvector containing the CMV promoter

Investigator: Bea

Comments: In order to obtain the CMV promoter in the correct RFC10 standard, oligos werde designed and ordered. For using the plasmid obtained from the iGEM team 2008, a retrafo has to be performed.

Re-transformation:

• Construct used: expressionvector + CMV (BBa_TV_CMV)
  • clone 7
  • clone 8
• concentration measureed with NanoDrop:
  • clone 7 (Bba_TV_VMV_7): c = 334, 65 ng/µL
  • clone 8 (Bba_TV_VMV_8): c = 183,77 ng/µL
• Plasmid need to be diluted in order to obatain the final 100pg.
  
• Dilution: 1:1000
• Volume needed of 1:1000 dilution of each clone:
  • clone 7 (Bba_TV_VMV_7): v = 0,29 µL
  • clone 8 (Bba_TV_VMV_8): v = 0,54 µL
• 100 µL aliquot of XL1-B were thawed on ice and divided into 50µL each. The proper amount of each clone was added.

Continuation: ITRs

Investigator: Hanna

In order to convert also the left ITR into RFC10 standard, the FANCY method was repeated with the newly arrived oligos (präfix and suffix).

Practical Cloning: Digestion of pAAV_MCS with AlwNI

• plasmid: P9 (270 ng/µL) = pAAV_MCS
• buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab:120) AlwNI

Comments: No.

Digestion

• Incubation: 1 h 20 minutes

Agarose-Gel:

0.5 g Agarose, 50 mL TAE (1 %), 3 µL GELRED, at 115 Volt, running time: 50 minutes

• Marker: GeneRuler ladder mix

Gelextraction

Gel measurement:

Practical Cloning: Digestion of left ITR fragment with NotI + PstI

• buffer used: 4 ; Restriction-enzymes used: Enzyme 1 NotI-HF; Enzyme 2 PstI-HF

Comments: Due to the digestion of an gelex- and very small fragment (ITR ~ 150 bp), the whole gelex-volume was used for digestion.

Digestion

• Incubation: 1 h 15 minutes

Agarose-Gel:

1.3 g Agarose, 65 mL TAE (2 %), 3 µL GELRED, at 115 Volt, running time: 45 minutes

• Marker: GeneRuler ladder mix

Gelextraction

Gel measurement:

Practical Cloning: Digestion of pGA14_Präfix-rITR with EcoRI + PstI

• buffer used: 4 ; Restriction-enzymes used: Enzyme 1 EcoRI-HF; Enzyme 2 PstI-HF

Comments: No.

Digestion

Plasmid: P63.1; c = 501.14 ng/µL

• Incubation: 1 h 15 minutes

Agarose-Gel:

1.3 g Agarose, 65 mL TAE (2 %), 3 µL GELRED, at 115 Volt, running time: 45 minutes

• Marker: GeneRuler ladder mix

Gelextraction

Gel measurement:

Gel picture of left ITR fragment and Präfix-rITR fragment:

Comment: The left ITR fragment was, as expected, nearly not detectable. Nevertheless it was cut out of the gel, because the same probleme occured during the first fancy-performance, which turned out to be successful!

Practical Cloning: Digestion of pGA14 with EcoRI + NotI

• plasmid: P61 (200 ng/µL) = pGA14_Cerulean
• buffer used: 4 ; Restriction-enzymes used: Enzyme 1 = EcoRI-HF; Enzyme 2 = NotI-HF

Comments: This digested vector used used for both ligation approaches.

Digestion

• Incubation: 1 h 15 minutes

Agarose-Gel:

0.5 g Agarose, 50 mL TAE (1 %), 3 µL GELRED, at 115 Volt, running time: 60 minutes

• Marker: GeneRuler ladder mix

Gelextraction

Gel measurement:

Hybridization of ITR-Oligos

left ITR:

• 10 µL Oligo 1 (O100 = lITR_Präfix_RFC10_for bzw. O104 = rITR_Suffix_RFC10_for)
• 10 µL Oligo 2 (O101 = lITR_Präfix_RFC10_rev bzw. O105 = rITR_Suffix_RFC10_rev)
• 4 µL 100 mM TrisHCl pH8
• 8 µL 5 mM MgCl2
• 8 µL H2O

Total: 40 mL

Because some of the oligos produce strong secondary structures, we used the ORIGAMI1 program:
1) initial denaturation: 99°C, 7 minutes
2) 99°C, 1 minute
3) 73 x repetition of 2) -> -1°C, R = 0.3 °C/sec
4) hold 4°C

Ligation

1. Ligation of left ITR + Präfix + pGA14:

• Präfix-Oligos: 1 µL
• left ITR: 8 µL
• pGA14: 8 µL
• Buffer: 2 µL
• T4 Ligase: 1 µL

Total: 20 µL

2. Ligation of Präfix-rITR + Suffix+ pGA14:

• Präfix-Oligos: 1 µL
• Präfix-rITR: 8 µL
• pGA14: 8 µL
• Buffer: 2 µL
• T4 Ligase: 1 µL

Total: 20 µL

The ligation approaches were incubated at 16°C over night (thermocycler).

Biobrick of GMK

Investigators: Stefan

aim: Biobrick construction of pAAV_RFC25_gmk for further addition of TK30.
result: Transformation worked out, clones can be picked.
expected construct:

Digestion with XbaI and AgeI and ligate into vector which was digested with XbaI and AgeI as well.

PCR of gmk:

• DNA template: puB6_V5_His6_clone1 + TK/GMK; concentration: 493,7 ng/µl

• PCR program: GMK

Digestion of pAAV_RCF25:

• DNA-Concentration: 321,5 ng/µl

Gel running of digested vector and PCR products

• 1% Agarose gel
• 3 µl Gelred, 8 µl DNA-Ladder-Mix
• 125 Volt, running time: 60 minutes

Sample of Hanna was loaded as well, see extra journal entry.

Gelextraction

• Gel extraction performed following standard protocol provided by Qiagen

Digestion of PCR products

Purification of PCR products, Ligation, Transformation

Purification has been followed by a different protocol (see Sven); has to be added in the standard protocols !

• Measure DNA-concentration with Nanodrop:

c(pAAV_RFC25) = 35,46 ng/µL
c(gmk) = 72,84 ng/µL

• Calculation of volume needed for ligation:

c(pAAV_RFC25) = 7,56 µL
c(gmk) = 1,44 µL

71. Labortag 27.7.2010

Cloning of linkers in pSB1C3_RFC25_CFP1.1

Investigator: Jessica
Linkers longlinker, SEG and GSAT should be cloned into the RFC25-standard

• P66: GSAT_linker_pMA 242,7 ng/µl
• P67: LonglinkerA50_pMA 230,4 ng/µl
• P71: SEG_pMA 211,8 ng/µl
• P51: pSB1C3_RFC25_CFP1.1 230,1 ng/µl

all linkers and pSB1C3_RFC25_CFP1.1 are digested with AgeI and XbaI for 2 hours

• 1% Agarose gel
• 3 µl Gelred, µl DNA-Ladder-Mix for P51, Low range for P66,67,71
• 130 Volt, running time: 45 minutes for P51, 90 Volt, running time 30 minutes for P66,67,71

• weight of P66 gel extract: 0,04g
• weight of P67 gel extract: 0,09g
• weight of P71 gel extract: 0,1g
• weight of P51 gel extract: 0,15g

finally called:

• pSB1C3_RFC25_longlinker P96
• pSB1C3_RFC25_GSAT P97
• pSB1C3_RFC25_SEG P98

Nanodrop

• Longlinker (P67): 2,1ng/µl
• SEG (P71): 3,7ng/µl
• GSAT (P66): 1,4ng/µl
• pSB1C3_RFC25: 29,6ng/µl
  

Ligation

• P51:P66 - 2,2µl:6,8µl
• P51:P67 - 2,53µl:6,47µl
• P51:P71 - 5,54µl:3,46µl
  

Transformation

• 4µl DNA was added to 50µl XL1b

Result sequencing of pAAV_RFC25_mGMK_TK30

Investigator: Bea

Comments: Results of the PCR and cloning experiment from lab day: 23.07.2010 (Mini-Prep). The sucessful insertion of mGMK_ZK 30 into the vector pAAV_RFC25 can be confirmed and is ready for cell culture.

Results sequencing of plasmid P81 (pAAV_RFC25_mGMK_TK30 clone 1)

Results sequencing of plasmid P82 (pAAV_RFC25_mGMK_TK30 clone 2)

Results sequencing of plasmid P83 (pAAV_RFC25_mGMK_TK30 clone 3)

Quickchange @ Pst in RepX 68 and RepX 78

Investigator: Kira

PCR program: PstI

• The PCR-products were digested with 0.5 µl DpnI
• Transformation was performed according to the standard protocol in XL1B cells

Site-directed mutagenesis of pAAV_RC_PstI_310+4073 (pAAV_RC_1.1) with BamHI and SalI

Investigator: Adrian, Bea

Comments: Site-directed mutagenesis was perfomed with pAAV_RC_PstI_310+4073 (pAAV_RC_1.1) in order to delete BamHI and SalI in the plasmid. These restriciton sites need to be removed because they are the single-cutter in the loops which will be used for targeting.

Protocol: Different PCR programs were used for performing the Quickchange site-directed mutagenesis protocol.

1. Longer elongation/extension time
2. Shorter elongation/extension time

1. PCR reaction with PCR program with longer elongation time:

DNA template used: pAAV_RC_PstI_310+4073 (pAAV_RC_1.1) = P87
A dilution of 1:20 was prepared and it was calculated the amount of DNA template needed for each site-directed mutagenesis reaction.

Used PCR programs for both SDMs:

Comment: Program has a long extension time because Quikchange Kit (Stratagene) recommends 1 minute per kb. Our vector has a length of 7300 bp therefore 7 minutes and 30 seconds were chosen for proper elongation of the whole vector.

2. PCR reaction with PCR program with shorter elongation time:

DNA template used: pAAV_RC_PstI_310+4073 (pAAV_RC_1.1) = P87
A dilution of 1:20 was prepared and it was calculated the amount of DNA template needed for each site-directed mutagenesis reaction.

Used PCR programs for both SDMs:

Comment: Quikchange Protocol (Stratagene) recommends 30 seconds per kb chosing a elongation temperature of 68°C. Our vector has a length of 7300 bp therefore 4 minutes were chosen for proper elongation of the whole vector. In order to see wheter this PCR program works aswell the same PCR ingredients were chosen as it can be seen in the table above.

Summary: Four site-directed mutagenesis have been performed with the plasmid pAAV_RC_PstI_310+4073 (pAAV_RC_1.1) = P87.

• Site-directed mutagenesis in order to delete restriction site BamHI
  • with shorter elongation time (modified protocol)
  • with longer elongation time
• Site-directed mutagenesis in order to delete restriction site SalI
  • with shorter elongation time (modified protocol)
  • with longer elongation time
• The samples were digested with 0,5µL DpnI after site-directed mutagenesis were performed in order to remove bacterial DNA template.
• Trafos have been performed with all four samples. Trafo plates are containing ampicillin.

Cell culture

Investigator: Adrian

• 3 x 6er dishes for Transduction were seeded and passaged(P14)

ITRs: Trafo of overnight-ligation approaches

Investigator: Hanna

Comments: Cloning approach was continued: RFC10-Präfix_rightITR was ligated to RFC10-Suffix and pGA14 and leftITR was ligated to RFC10-Präfix and pGA14 over night. Trafo of these samples was performed in order to pick clones tomorrow.

Trafo was performed following the standard protocol. Abberance: 4 µL DNA was added to BL21 cells.

PCR: beta-Globin

Investigator: Hanna
Because sequencing delivered, that cloning of the beta-globin intron into pSB1C3 didn't work (Cerulean was still in the vector), the PCR was performed one more time:

PCR program:

• 98°C, 1'

• 98°C, 30
• 63°C, 25
• 72°C, 15

-> 8x

• 98°C, 30
• 72°C, 20

-> 20x

• 1 x 72°C, 5'
• Hold: 4°C

Repetition of PCR for hGH BioBrick Production

Investigator: Bea

Comments: Sequencing of pSB1C3_hGH did not reveal any insertion of hGH into pSB1C3. Instead of hGH CFP was found in the vector indicating that undigested or partially digested vector religated. Therefore a new pCR for generating the BioBrick was performed over night.

PCR reaction:

Used PCR programs:

Comment: PCR will be performed over night.

Picking clones from pAAV_RFC25_mGMK

Investigator: Bea

Comment: Trafo plate looked good. On control plate only few clones grown in contrary to the XL1-B{pAAV_RFC25-mGMK} plate. Two clones were picked.

• 10 mL containing 10µL ampiciliin were inoculated.
• Put into 37°C room on shaker.

Picking clones from Retrafo of pTV_CMV clone 7 and clone 8

Investigator: Bea

Comment: Trafo plates looked good. Enough clones grown. Two clones of each plate were picked

• 10 mL containing 10µL ampiciliin were inoculated with each clone respectively.
• Put into 37°C room on shaker.

72. Labortag 28.7.2010

Results Trafo pAAV_RC_1.1_BamHI and pAAV_RC_1.1_SalI

Investigator: Bea

Results: Too many bacteria grown resulting in a bacterial lawn. Possible reasons could be some problems with the new prepared ampicillin because trafo from Hanna containing the ITRs had the same results. Trouble shooting done by Hanna.

Proceed with BioBrick Production of hGH and beta-globin

Investigator: Bea

Comments: PCR was started yesterday and conducted over night.

Proceeding:

• Prepare 1% agarose gel and load samples on gel.
• Run time: 45 minutes

Result of gel:

Marker did not resolute very good. Expected sizes of PCR products:

• hGH = 510 bp
• beta globin intron: 524 bp

It can be seen (or estimated) that two intensive bands are visible at around 500 bp. Therefore these two bands were cut out and a gel extraction was performed. The lower visible band at around 100 bp of hGH is an additional band generated at the pCR reaction. The two intensive blurred bands belong to the loading dye added to the samples.

After gel extraction was performed, the PCR prdoucts hGH and beta-globin were digested:

DIGESTION of PCR products

• Incubate for 2h at 37°C
• Perform PCR purification following standard protocols
• Elute in 20µL EB-buffer, put on 50°C for 3 minutes before centrifugating
• Measure DNA with NanoDrop:
  • c(hGH) = 58,14 ng/µL
  • c(ß-globin) = 83,10ng/µL

In the meantime prepare the digestion of vector pSB1C3_CFP (P51)

DIGESTION of pSB1C3_CFP

• Incubate for 2h at 37°C
• Load sample on 1% preparative agarose gel
• Run for 45 minutes at 110 V

Result of gel:

The vector was digested with XbaI and PstI. Expected sizes have been:

• pSB1C3 = 2072bp
• CFP: 732bp

It can be seen that the vector pSB1C3_RFC25_CFP was digested and two visible bands can be seen on the gel. The lower band corresponds to the CFP fragment (~700 - 800 bp), the higher band corresponds to the plasmid backbone at around 2000-3000 bp. The higher band was cut out and extracted by gel extraction.

• Perform gel extraction and measure DNA with NanoDrop
  • c(psb1C3) = 29,52 ng/µL

LIGATION of pSB1C3 with hGH and beta-globin

Calculated ratio of pSB1C3 (vector) and hgh(insert) or betaglobin (insert)