Team:LMU-Munich/Notebook/Apoptosis

ApoControl Notebook

What we did

Short description of our work, our results and our supporters

The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.

Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.

The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.

In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.

We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.

If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.

Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.

The protocols we used are listed here: Protocols

These Biobricks we submitted:

• BBa_K368004: attP+eGFP+SV40PA
• BBa_K368011: eGFP+SV40PA
• BBa_K368016: TEVrecognition site+N-degron+SF3b155
• BBa_K368019: TEV-Protease+p14*+TEVrecognition site

Sources, helpers and supporters:

• Prof. Dr. Angelika Böttger :
  • prevTRE (tet-on CMV promoter; inducible by doxycycline in special cell lines)
  • supported the construction ideas and would have given us the cells and mediums we would have needed
  • SV40PA (Polyadenylation site): gave us a vector containing it
  • Human Bak: her assistant Erika Clement gave us appropriate cDNA

• Dr. Arnim Weber: submitted us a vector with human Bak
• Dr. Philipe Soriano: (Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007))
  • Sequences of attB and attP site
  • PhiC31o was bought via addgene
• Knop, M (Heidelberg): (Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))
  • TEVrecognition site+N-degron+SF3b155
  • TEV-Protease+p14*+TEVrecognition site
• Prof. Dr. Thorsten Mascher:
  • Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
• Prof. Dr. Kirsten Jung:
  • Helped with ideas and fundraising
• Dr. Susanne Gebhard:
  • Helped with trouble-shooting and materials
• Prof. Dr. Andreas Brachmann:
  • Sequenced our samples
• Partsregistry:
  • eGFP (BBa_I714891)
  • CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
  • ccdB amp, cam, tet, kan in E.coli DH3

Contents>

8-10-2010

Transforming competent cells

- eGFP Biobrick: BBa_I714891 SDY_eGFP (Kanamycin)

- TEV recogn N Degron SF3 = pDS7 (Ampicillin)

- TEV p14 recogn = 190-6 (Ampicillin)

-> Protocol: (3 Transformation)

- We added 2 µl DNA

- We plated out 200 µl

Plasmid Isolation

- CMV-Promoter Biobrick: BBa_J52034

-> Protocol:(4 Plasmid extraction from cells)

- Prepared overnight culture, measured concentration of DNA

-> Poor results -> thrown away

8-11-2010

New Plasmid Extraction

- CMV-Promoter Biobrick: BBa_J52034

-> Protocol: (4 Plasmid extraction from cells)

- Plasmid concentration: 143ng/µl

Prepared overnight culture of eGFP BBa_I714891

- 3 ml LB-Media + 4 µl Kanamycin

- Inoculated with 1 colony of BBa_I714891 -> 37°C

Prepared overnight culture of 190-6 and pDS7 and eGFP (BBa_I714891) in falcons

- for 190-6 and pDS7: 10µl Ampicillin + 10 ml LB-Media + colony of plate

- for eGFP: 13,3 µl Kanamycin + 10 ml LB-Media + 1 colony of plate

Restriction digestion of CMV-Promoter BBa_J52034 with EcoRI and PstI

-> mixed

- plus: EcoRI (10µg/µl): 0,5 µl resp. PstI (10µg/µl): 0,5 µl

- incubated at room temperature from 12:10 to 15:00, 1 hour at 37°C, 2 hours at 60°C

- frozen at -20°C

Prepare new/fresh overnight culture of CMV-Promoter Biobrick: BBa_J52034

- 1 ml of "old" culture + 3 ml LB-Media + 4 µl Kanamycin -> 37°C

8-12-2010

Plasmid Extraction of pDS7, eGFP, 190-6

-> Protocol: (4 Plasmid extraktion from cells)

- pDS7 (458ng/µl), eGFP (55ng/µl), 190-6 (193ng/µl)

Restriction digest of pDS7, eGFP, 190-6

- with EcoRI and PstI in buffer H (for testing DNA is correct)

-> Protocol: (5 Restriction digest)

- 10µg DNA: pDS7 (2µl), eGFP (15µl), 190-6 (10µl)

Plate colonies for plasmid extraction

- CMV (Kanamycin), eGFP (Kanamycin), pDS7 (Ampicillin), 190-6 (Ampicillin))

- PhiC31o plated on Ampicillin-Agar, stored at 37°C

50% Glycerol made

- for PhiC31o glycerol stock (produced later)

8-13-2010

Gelfoto from the EcoR1 and Pst1 Restrictiondigest of 190-6, eGFP, pDS7 and CMV

Inoculate CMV into LB medium with ampicillin

- CMV (BBa_J52034) from 10.8.2010 inoculated into LB medium with ampicillin, as falsly inoculated in Kanamycin

Agarosegelelectrophoresis with digestions

->Protocol (11 Agarose gel electrophoresis)

- Agarosegelelectrophoresis with the digestions (CMV, eGFP, pDS7, 190-6), 125V for 30 minutes and then for 20 minutes;

- expected DNA bands: 190-6 (4840bp, 1903bp), pDS7 (8027bp, 6bp), CMV (654 bp (Insert), 2079bp (Plasmid)), eGFP (720bp (Insert), 2750bp (Plasmid))

- Correct DNA bands for 190-6 (~4800bp, ~1900bp, ~6700bp (undigested plasmid)) and eGFP (~2000bp (Plasmid), ~750 bp (Insert)); CMV probably not digested (two bands; one probably normal, one supercoiled) and pDS7 not clear

Restriction digest from CMV and pDS7

-> Protocol (5 Restriction digest)

- Restriction digest from CMV (EcoR1, Pst1; 6µl DNA, buffer H) and pDS7 (EcoR1, Spe1; 2µl DNA, buffer B)

Agarosegelectrophoresis with digestions

->Protocol (11 Agarose gel electrophoresis)

- Agarosegelelectorphoresis for 30 minutes, 150V

- Expected DNA bands: CMV see above, pDS7 (3647bp, 3369bp, 1011bp, 6bp)

- false DNA bands CMV (~1200 bp, ~2000 bp) and pDS7 (~8000bp two bands, ~1100 bp); required to isolate a new colony for these two Plasmidextractions

Plated CMV on Ampicllin-Agar

- Plated the colony from CMV (BBa_J52034) for Plasmidextraction (Ampicillin), as falsly plated on Kanamycin

8-14-2010

weekend

8-15-2010

weekend

8-16-2010

Planting colonies

- transfer 1 ml PhiC31o culture to new LB medium + Amp, 37°C

- pick up CMV and pDS7 colonies from plates and transfer to LB medium+Amp, 37°C

Plasmid Extraction of PhiC31o

->Protocol (4 Plasmid extraktion from cells)

- plasmid extraction of PhiC310

->27,5ng/µl DNA and second plasmid extraction of PhiC310 (i. o. to get more DNA); first eluation-step with first eluation-extraction

-> 60ng/µl DNA

Restriction digest

->Protocol (5 Restriction digest)

- restriction digest of PhiC310 with EcoR1 and Spe1

restriction digest in the thermo cycler (program "Verdau", see protocol)

Handling primers after arrival (1,2,3,4,5,6,11,12)

->Protocol (9 Handling primers)

PCR preparations

- 10mM dNTP mix made from 100 mM dATP, dGTP, dCTP, dTTP by taking 100µl of each and adding 600µl H ₂ O

PCR 1 and 6

- PCR of the tet inducible CMV minimal promotor out of prevTRE (=PCR 1 with Primer 1 and 2) and SV40PA out of pcDNA3 (=PCR 6 with Primer 11 and 12)

->Protocol (10 PCR with Pfu)

Mixture:

Programme:

Glycerolstock of PhiC31o

- Glycerolstock of the colony of PhiC31o for the plasmidextraction

8-17-2010

Agarose gel electrophoresis of PCR6 which shows that PCR6 is about 200bp

Plate CMV and pDS7 colonies on Ampicillin-Agar

- colonies for plasmidextraction of CMV and pDS7 plated on Ampicillinplates

Plasmid Extraction of CMV and pDS7

- plasmidextraction of CMV (2,5ng/µl) and pDS7 (10ng/µl) the A260/A280 value was 1.333, which means that it was 90% Protein and only 10% DNA (should be 1,8); new plasmidextraction needed

new overnight cultures of CMV and pDS7 for a new plasmidextraction made

Agarose gel electrophoresis

-> Protocol (11 Agarose gel electrophoresis)

- Agarose gel electrophoresis of the restriction digest of PhiC31o and PCR 1 and 6

- the right bands found for PhiC31o (~2900,~2400,~250)

- the right band found for PCR1 (~450)

- no band found for PCR6; new electrophoresis needed with more DNA loaded

Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6	Agarose gel electrophoresis of (from left to right) PhiC31o, PCR1 and PCR6 which shows that PCR1 is between 250 and 500 bp

- new agarose gel electrophoresis from PCR6 with 5µl DNA instead of 3µl (image not yet shown)

- the right band found for PCR6 (~200)

New overnight cultures of CMV and pDS7

- the overnight colonies didn't grow; new colonies (CMV and pDS7) picked from plate and inoculated in LB Ampicillin

PCR purification of PCR 1 and 6

-> Protocol (12 Gel extraction or PCR Clean up)

- DNA concentration of the PCR 1 and 6 products measured: PCR1: 410ng/µl (A260/A280=1.253) PCR6: 568ng/µl (A260/A280=1.275)

- PCR Purification with Promega Kit

-> PCR1: 230ng/µl (A260/A280=1.769)

-> PCR6: 37.5ng/µl (A260/A280=1.667)

8-18-2010

Agarose gel electrophoresis of (from left to right) CMV and pDS7 showing the right bands for pDS7

Plasmid Extraction of CMV and pDS7

-> Protocol (4 Plasmid extraction from cells)

- Plasmid extraction of CMV (97.5ng/µl; A260/A280=1.857) and pDS7 (212ng/µl; A260/A280=1.848)

Restriction digestion

-> Protocol (5 Restriction digestion)

- Restriction digestion of CMV (EcoR1 + Pst1; 10µl DNA, buffer H) and pDS7 (EcoR1 + Spe1; 5µl DNA, buffer B)

-> expected DNA bands: CMV: 2079bp (plasmid) + 654bp (Insert); pDS7: 7022bp + 1011bp

Agarose Gel electrophoresis of digested CMV and pDS7

-> Protocol (11 Agarorse gel electrophoresis)

-> right DNA bands for pDS7 (~7000bp, ~1000bp)

-> false DNA bands for CMV

- Starting PCR 2a and 2b (replication and mutagenesis of pDS7): 3 µl DNA and 50°C Annealing Temperatur (other same as 8-16-2010)

8-19-2010

Agarose gel electrophoresis of PCR 2a and 2b

-> Protocol (11 Agarose gel electrophoresis) (150V, 30min)

Agarose gel electrophoresis of (from left to right) PCR2a and PCR2b

-> the right bands for PCR2a (~300bp) and PCR2b (~700bp)

- New agarose gel electrophoresis with all of the PCR product for gel extraction (150V, 30min)

Gel extraction of the DNA from PCR2a and PCR2b

-> Protocol (12 Gel extraction or PCR Clean up)

- DNA concentration measured; problem with nanodrop as too low concentration; lyophille used to reduce volume

- DNA concentration measured again: PCR2a: 70ng/µl A260/A280=1.647; PCR2b: 45ng/µl A260/A280=1.5

PCR 3 (joining PCR of 2a and 2b)

- PCR3 (the joining PCR of PCR2a and 2b; Joining of the TEVrecogn-N-Degron-SF3 part) done: 1.3 µl of PCR2a and 4.7 µl of PCR2b makes 300ng of a 1:1 solution of both to be joined DNA parts. Annealing temperature: 50°C

-> Protocol (10 PCR with Pfu)

8-20-2010

Agarose gel electrophoresis of PCR3

left column: marker; rightmost column: PCR3

-> Protocol: 11 Agarose gel electrophoresis (150V, 30min)

-> expected band: ~1000bp

-> false band: ~500bp

- probable reason: mini photometre was influenced by gel extraction chemicals, therefore it measured false DNA concentrations and false template masses were calculated

-> New 2a and 2b PCR

New PCR (2a and 2b)

-> Protocol: 10 PCR with Pfu

(see 8-18-2010, but 35,5µl water)

8-21-2010

weekend

8-22-2010

weekend

8-23-2010

Agarose gel electrophoresis of PCR 2b

-> Protocol: 11 Agarose gel electrophoresis

- expected band: 700bp

-> no band shown on gel -> new PCR 2b

PCR 2b

- start PCR 2b with PCR 2b from 8-13-10 as template ( 1:20 and 1:100 diluted; 1µl)

-> Protocol: 10 PCR with Pfu

- annealing temperature: 50°C; amount of water: 37,5µl

Agarose gel electrophoresis of PCR 2b 1:20 and 1:100

-> Protocol: 11 Agarose gel electrophoresis

- expected bands: each ~ 700bp

- false bands: ~ 200bp

-> new PCR with 2ng, 5ng, 10ng template pDS7

PCR 2b with 2ng, 5ng, 10ng template pDS7

- pDS7 1:100 diluted(-> 2,1 ng/µl)

Mixture:

-> Protocol: 10 PCR with Pfu

PCR 2a gel extraction

- Quiagen kit (QuiaexII)

-> Protocol: 14 QIAEX II gel extraction

Start 3 CMV overnight cultures

8-24-2010

agarose gel electrophoresis of PCR 2b

-> Protocol: 11 Agarose gel electrophoresis

Agarose gel electrophoresis of (from left to right) PCR2b (2ng (cut out), 10ng, 5ng template) showing the right bands for 2ng, 5ng template

- expected bands: right bands with 2ng and 5ng template (~700bp), no band with 10ng template

CMV plasmid extraction

-> Protocol: 4 Plasmid extraction from cells

Plasmid extractionof 3 different overnight cultures.

- results:

• 52,5 ng/µl A260/A280= 1.312
• 133 ng/µl A260/A280= 1.710
• 80 ng/µl A260/A280= 1.600

CMV restriction digestion

-> Protocol: 5 Restriction digestion

- CMV restriction digest: EcoRI, PstI, buffer H

• 19µl, H₂O : 0µl
• 6µl, H₂O : 9.5µl
• 12.5µl, H₂O : 3µl

PCR 2b gel extraction

- PCR2b was gel extracted (with Qiagen gel extraction kit), 17.5 ng/µl a260/A280= 1.750

-> Protocol: 14 QIAEX II gel extraction

PCR 3 (fusion of 2a and 2b)

- PCR3: conducted again at 52°C annealing temperature

• 10.5 ng (from PCR2b) 0.6µl

• 4.5 ng (from PCR2a) 0.9µl (1:10 diluted)

PCR2a	0.9 µl
PCR2b	0.6 µl
primer3	2.5 µl
primer6	2.5 µl
dNTPs	1 µl
Pfu	0.5 µl
10xbuffer	5 µl
H2O	37 µl

-> Protocol: 10 PCR with Pfu

agarose gel electrophoresis of CMV digestion

- agarose gel electrophoresis (150V, 25 min) of the CMV digestion

-> bands are wrong again ( ~ 1200bp, 2000bp)

8-25-2010

Agarose gel electrophorese of PCR 3

-> Protocol: 11 Agarose gel electrophoresis

- expected band: ~1000bp

- false band: ~400bp

Plasmid extraction of ccdB tet and ccdB strep

Plasmid extraction of pSB1C3 with BBa_P1010

-> Protocol: 4 Plasmid extraction from cells

- results:

Plate ccdB amp, cam, tet

- plate ccdB with ampicilline, chloramphenicol, tetracycline resistance on LB agar with appropiate antibiotic.

Overnight culture of ccdB kan

- Overnight culture of ccdB with kanamycine resistance in LB medium with kanamycine

PCR 7a, 7b, 9, 10

->Protocol: 10 PCR with Pfu

Mixture

Standard PCR; annealing temperature: 60°C

8-26-2010

Agarose gelelectrophoresis of PCR 7a, 7b, 9, 10

->Protocol: 11 Agarose gel electrophoresis

- 150V, 25min

Plasmid extraction of ccdB kan

-> Protocol: 4 Plasmid extraction from cells

-result: concentration: 25ng/µl; A260/A280= 2,0

New PCR 7a, 7b, 9, 10

Mixture

Program:

gradient PCR, 42-69°C annealing temp.

->Protocol: 10 PCR with Pfu

Overnight culture of ccdB amp, tet, cam

Inoculate one colony each in 5ml medium with approptraite antibiotic.

8-27-2010

Agarose gel electrophoresis of PCR 7a, 7b, 9, 10

->Protocol: 11 Agarose gel electrophoresis

150V, 25min, 75mA

from left to right: 7a, 7b, 9, 10, Marker

Plasmid extraktion of ccdB amp, tet, cam

->Protocol: 4 Plasmid extraction from cells

results:

New PCR 7a, 9

Mixture:

- 2ng template: see 26-8-10

- 4ng template: see 26-8-10, but 2µl template and 35,25µl water

-> Protocol: 10 PCR with Pfu

Restriction digestion of ccdB amp, kan, cam, tet

-> Protocol: 5 Restriction digestion

- only 90min 37°C incubation

- EcoRI, PstI, Buffer H

Agarose gelelectrophoresis of PCR 7a, 9, ccdB restriction digestion

150v, 25min, 75mA

-> Protocol: 11 Agarose gel electrophoresis

results:

- PCR7a, 9: false band at 200bp

- ccdB: each digestion leads to a right band with ~ 650bp

8-28-2010

weekend

8-29-2010

weekend

8-30-2010

New PCR 7a and 9

Mixture

-> Protocol: 10 PCR with Pfu

PCR program

PCR 7: Annealing Temperature 60°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time

PCR 9: Annealing Temperature 55°C - 25 x 1 min Annealing time and 5x 1,30 min Annealing time

Gel extraction of PCR 7b, 10

->Protocol: 14 QIAEX II gel extraction

results:

Agarose gel electrophoresis of new PCR 7a, 9

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

- results:

- 7a: no band shown

- 9: false band (~200bp)

New PCR 3

-> Protocol: 10 PCR with Pfu

PCR2a	0.9 µl
PCR2b	0.6 µl
dNTPs	1 µl
Pfu	0.5 µl
10xbuffer	5 µl
DMSO	1,25µl
H2O	36,75 µl
sum	45µl

- new method: standard PCR without primers (10 cycles, 56°C annealing temp.)

- then add 2,5µl of primer 3 and 6

- 30 cycles standard PCR (54°C annealing temp.)

8-31-2010

Gel photo of (left to right) PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu), PCR3(Phusion)

Agarose gel electrophoresis of PCR 4a, PCR4b, PCR3(Pfu), PCR3(Phusion)

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

- results:

- PCR4a(2.5ng template), PCR4a(5ng template),PCR4b(2.5ng template), PCR4b(5ng template), PCR3(Pfu): no band shown

- PCR3 (Phusion): right band (~1000bp)

New PCR PCR4a, PCR4b, PCR7a, PCR9

-> Protocol: 10 PCR with Pfu

PCR mixture for PCR4a, PCR4b

template	37.25 µl (200ng)
dNTPs	1 µl
Pfu	0.5 µl
10xbuffer	5 µl
DMSO	1,25µl
sum	50µl

Standard PCR program with annealing temperature PCR4a: 51.1°C, PCR4b: 48.5°C.

-> Protocol: 15 PCR with Phusion

PCR mixture for PCR7a, PCR9

template	4 µl (8ng)
dNTPs	1 µl
Phusion	0.5 µl
5xbuffer	10 µl
DMSO	1,25µl
H2O	28.25 µl
sum	50µl

PCR program: Phu62

98°C	1 min
98°C	10 sec
62°C	20 sec
73°C	30 sec
return to step 2 for 29 cycles
73°C	10 min
12°C	forever

Gel photo of (left to right) PCR3, ladder, PCR7a, empty, PCR9

Agarose gel electrophoresis of PCR 3, PCR7a, PCR9 for gel extraction

-> Protocol: 11 Agarose gel electrophoresis

120V, 30min

- results:

- PCR3; right band (~1000bp) and side-product

- PCR7a: no band

- PCR9: right band (~800bp) and side-product

Gel extraction of the DNA from PCR3 and PCR9

-> Protocol (12 Gel extraction or PCR Clean up)

results:

PCR 9: 22,5ng/µl; A260/A280=1,8

PCR 3: 22,5ng/µl; A260/A280=2,25

New PCR 4a, 4b, 7a with DreamTaq

-> Protocol: 16 PCR with DreamTaq

PCR mixture for PCR7a

template	5 µl (10ng)
dNTPs	5 µl
DreamTaq	0.33 µl
10xbuffer	5 µl
DMSO	1,25µl
H2O	28.5 µl
sum	50µl

Primers for PCR 7a: 13,14

Annealing temp: 60°C

PCR mixture for PCR4a,4b

template	36,5 µl (180ng HeLa cDNA)
dNTPs	5 µl
DreamTaq	0.33 µl
10xbuffer	5 µl
DMSO	1,25µl
sum	50µl

PCR 4a

Primers for PCR 4a: 7,8

Primers for PCR 4b: 9,10

Annealing temp: 50°C

PCR program:

95°C	1 min
95°C	30 sec
50/60°C	30 sec
72°C	1 min (1kb/min)
return to step 2 for 29 cycles
72°C	10 min
12°C	forever

9-01-2010

Agarose gel electrophoresis of PCR4a, PCR4b, PCR7 (DreamTaq)

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

results: no product

New PCR 4a, 4b with DreamTaq, Pfu, with concentration gradient and touch-down PCR

-> Protocol: 16 PCR with DreamTaq; 10 PCR with Pfu

PCR mixture for DreamTaq

Concentration	Low	Middle	High
template	5 µl (1:1000)	31.75µl (1:1000)	2µl (1:10)
dNTPs		5 µl
DreamTaq		0.33 µl
10xbuffer		5 µl
DMSO		1,25µl
H2O	26.75 µl	0	29.75
sum		50µl

PCR mixture for Pfu

Concentration	Low	Middle	High
template	5 µl (1:1000)	37.25µl (1:1000)	2µl (1:10)
dNTPs		1 µl
Pfu		0.5 µl
10xbuffer		5 µl
DMSO		1,25µl
H2O	32.25 µl	0	35.25 µl
sum		50µl

Primers for PCR 4a: 7,8; PCR 4b: 9,10

-> Protocol: Thermal cycler program: Touch down

Agarose gel electrophoresis of PCR4a, PCR4b

-> Protocol: 11 Agarose gel electrophoresis

150V, 25min

from left to right: 4a: P1, P2, P3, D1, D2, D3; 4b: P1, P2, P3, D1, D2, D3

key:

"P"= PCR with Pfu

"D"= PCR with DreamTaq

"1"= low template concentration

"2"= middle template concentration

"3"= high template concentration

expected bands:

• 4a: 330bp -> P2 and P3 show right bands and "primer clouds"(?)

• 4b: 376bp -> P1, P2, P3 show right bands and "primer clouds" (?)

9-02-2010

Agarose gel electrophorese of PCR 4a P2, 4b P2

-> Protocol: 11 Agarose gel electrophoresis

- 120V, 45min, 1,5% Agarose gel

Agarose gel electrophoresis of (from left to right) PCR4aP2, Marker and PCR4bP2

- Cut out bands at ~~ 350bp and extract

PCR Agarose gel extraction

-> Protocol: 14 QIAEX II gel extraction

results:

New PCR 7a with Pfu and Phusion

template: 190-6, Primer 13,14

Mixture with Pfu

-> Protocol: 10 PCR with Pfu

PCR mixture with Phusion

template (~2ng)	1,0 µl
dNTPs	1 µl
Phusion	0.5 µl
5xbuffer	10 µl
DMSO	1,25µl
H2O	31,25µl
sum	50µl

-> Protocol: 15 PCR with Phusion

New PCR 4a, 4b with Pfu

-> Protocol: 10 PCR with Pfu

-> Protocol: Thermal cycler program: Touch down

Mixture see 9-1-10, twice 4a and 4b

Agarose gel electrophorese of PCR 4a, 4b, 7a, gel extracted 4a and 4b

-> Protocol: 11 Agarose gel electrophoresis

- 25min, 150V

from left to right: 4a*, 4a, 4b*, 4b, 7a Phusion, 7a Pfu, Ladder

-> result: 4b, 4b*: right bands (~330bp)

-remain: false bands/no band

from left to right: ladder, 4 columns pathway, 4a gelextr., 4b gelextr.

-> results: slight right bands for 4a and 4b, no "primer clouds" anymore.

9-03-2010

Overlapping PCR 5 with Pfu and Phusion

template: 4a, 4b Primer 7,10

Mixture with Pfu

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Annealing temperature: 54°C

PCR mixture with Phusion

PCR4a (~15ng)	1.2µl
PCR4b (~15ng)	2µl (1:10)
dNTPs	1 µl
Phusion	0.5 µl
5xbuffer	10 µl
DMSO	1,25µl
H2O	29.05µl
sum	50µl

-> Protocol: 15 PCR with Phusion

PCR program: standard PCR program for Phusion, Annealing temperature: 58°C

New PCR7a with Pfu

template: 190-6; Primer: 13,14

Mixture with Pfu

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Gradient: 54.4°C, 57.8°C, 61.4°C, 65.0°C

-> results:

-7a: only "primer cluods"

-5 Pfu: no defined product (slurred?)

-5 Phusion: "primer clouds"

9-04-2010

weekend

9-05-2010

weekend

9-06-2010

charges for sequencing

every charge 7µl

New overlapping PCR 5 with Pfu

template: 4a, 4b; 15ng

Primer 7,10

Mixture

-> Protocol: 10 PCR with Pfu

PCR program: standard PCR program for Pfu, Annealing temperature: 46.1°C,48.5°C, 51.1°C

-> results: "primer clouds"

New PCR 7a with Pfu new dilution of 190-6; 1:100