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

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:

• 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

Investigators: Anissa, Kerstin, Anna

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

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

56.Labortag 12.07.2010: ITR BioBrick production

Practical Cloning:

• experiment date: 12.07.2010 ; time: 11.00
• name of investigator: Anna, Kerstin, Anissa
• 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

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

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

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

Invetigators: 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

• experiment date: 13.07.2010 ; time: 16:00
• name of investigator: Bea
  • 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: Seed HEK, DAAD Timetable, Summary, Ordering ITR+TKGMKI, Quickchange for the PstI restriction sites in pAAV-RC

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)

Invetigators: 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 HEK

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

Summary

Investigators:

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

New millipore H2O, LB + Agar and DMEM medium were prepared
Investigators: Stefan, Patrick

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

TRAFO EVALUATION (PstI Quickchange Trafo from 14.07)
Investigator: Kira
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

• experiment date: 15.07.2010 ; time:10.00
• name of investigator: Kerstin, Anissa
• 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 sequenzed

• 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

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 Method

Trafo

Hanna

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

right ITR

Hanna

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: left ITR

Picking clones of pGA14_Präfix-lITR 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:

Calculation of ganciclovir cymeven concentration

Investigator Adrian:

• 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.

Investigators: Chris, Patrick

Test digestion of pAAV_RFC_mgmk_tk30: P54, P55, P56, P57
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. Investigators: Patrick, Chris

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

Investigator: 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

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

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