Apoptosis Notebook
Contents
8-02-2010
8-03-2010
Some test text in bold
We created following tests:
- test4
- test5
8-04-2010
Example of a table
| header 1
| header 2
| header 3
|
| row 1, cell 1
| row 1, cell 2
| row 1, cell 3
|
| row 2, cell 1
| row 2, cell 2
| row 2, cell 3
|
8-05-2010
this too is a table:
| H2Oddes
| 10,3 µl
|
| RE10 + Buffer H
| 2,0 µl
|
| acetylated BSA
| 0,2 µl
|
| DNA
| 6,0 µl
|
table with 3 cells
| apple | banana | peaches
|
| green | yellow | red
|
8-06-2010
text
8-07-2010
text
8-08-2010
test
test
8-09-2010
text Knallroter Text
farbnummern für farbige schrift: http://html.nicole-wellinger.ch/hilfen/farbenverzeichnis.html
test grüner text
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 iangeimpft) 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
| H2Oddest, sterile
| 10,3 µl
|
| RE10 + Buffer H
| 2,0 µl
|
| acetylated BSA (18ng/µl)
| 0,2 µl
|
| DNA (0,143µg/µl)
| 6,0 µl
|
-> 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 extrction
- 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 amicillin
- 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
| H2Oddest, sterile
| 0 µl
|
| Buffer B
| 2,0 µl
|
| BSA (1:10)
| 2 µl
|
| DNA (0,06µg/µl)
| 15,0 µl
|
| EcoR1
| 0,5 µl
|
| Spe1
| 0,5µl
|
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:
|
| pTRERev (0,15µg/µl)
| pcDNA3 (0,6 µg/µl)
|
| Primer
| 2*2,5µl (P1+P2)
| " (P11+P12)
|
| 300ng template
| 0,5µl
| 2µl
|
| 10x Buffer Pfu
| 5µl
| "
|
| dNTP Mix
| 1µl
| "
|
| Pfu Polymerase (3u/µl)
| 0,5µl
| "
|
| H2O
| 40,5µl
| 39µl
|
| summ
| 52,5µl
| 52,5µl
|
Programme:
| Denaturation
| 95°C
| 2min
|
| 30 times:
| Denaturation
| 95°C
| 1min
|
|
| Annealing
| 45°C
| 30sec
|
|
| Extension
| 73°C
| 2min
|
| Final Extension
| 73°C
| 5min
|
| Soak (end)
| 12°C
| infinite
|
Glycerolstock of PhiC31o
- Glycerolstock of the colony of PhiC31o for the plasmidextraction
| bacterial culture
| 800µl
|
| Glycerol (50%)
| 500µl
|
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; most right 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:
|
| 2ng
| 5ng
| 10ng
|
| Primer
| 2*2,5µl (P5+P6)
| 2*2,5µl (P5+P6)
| 2*2,5µl (P5+P6)
|
| 10x Buffer Pfu
| 5µl
| 5µl
| 5µl
|
| dNTP Mix
| 1µl
| 1µl
| 1µl
|
| template
| pDS7 (dil.)
| 1µl
| 2,5µl
| 5µl
|
| Pfu Polymerase (3u/µl)
| 0,5µl
| 0,5µl
| 0,5µl
|
| DMSO
| 1,25µl
| 1,25µl
| 1,25µl
|
| H2O
| 33,25µl
| 30,25µl
| 25,25µl
|
| sum
|
|
|
|
-> Protocol: 10 PCR with Pfu
PCR 2a gel extraction
- Quaigen kit (QuaiexII)
-> 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) CMV (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:
| ccdB tet:
| 50ng/µl;
| A260/A280= 1,818
|
Plate ccdB amp, cam, tet
- plate ccdB with ampicilline, chloramphenicol, tetracycline resistence on LB agar with appropiate antibiotic.
Overnight culture of ccdB kan
- Overnight culture of ccdB with kanamycine resistence in LB medium with kanamycine
PCR 7a, 7b, 9, 10
->Protocol: 10 PCR with Pfu
| PCR nr.
| template
| concentration
| dilution
| primer
|
| 7a
| 190-6
| ~200ng/µl
| 1:100
| 13,14
|
| 7b
| 190-6
| ~200ng/µl
| 1:100
| 15,16
|
| 9
| eGFP
| 55ng/µl
| 1:25
| 20,21
|
| 10
| PhiC31o
| 20ng/µl
| 1:10
| 22,23
|
Mixture
| template (~2ng)
| 1µl
|
| Pfu
| 0,5µl
|
| Primer *2
| 2,5µl *2
|
| 10x buffer
| 5µl
|
| dNTP Mix
| 1µl
|
| H2O
| 37,5µl
|
| sum
| 50µl
|
Standard PCR; annealing temperature: 60°C
8-26-2010
Agarose gelelectrophoresis of PCR 7a, 7b, 9, 10
->Protocol: 11 Agarose gel electrophoresis
- 150V, 25min
| PCR nr.
| expected bands
| result
|
| 7a
| 850bp
| no band
|
| 7b
| 402bp
| false band (200bp)
|
| 9
| 808bp
| no band
|
| 10
| 1888bp
| no band
|
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
| template (~2ng)
| 1µl
|
| Pfu
| 0,5µl
|
| Primer *2
| 2,5µl *2
|
| 10x buffer
| 5µl
|
| dNTP Mix
| 1µl
|
| DMSO
| 1,25µl
|
| H2O
| 36,25µl
|
| sum
| 50µl
|
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
| PCR nr.
| expected bands
| result
|
| 7a
| 850bp
| no band
|
| 7b
| 402bp
| right band (~400bp)+ false band (~150bp)
|
| 9
| 808bp
| false band (~200bp)
|
| 10
| 1888bp
| right band (~1900bp)+false band (~500bp)
|
Plasmid extraktion of ccdB amp, tet, cam
->Protocol: 4 Plasmid extraction from cells
results:
| Plasmid
| concentration
| A260/A280
|
| ccdB amp
| 57,5 ng/µl
| 1,917
|
| ccdB cam
| 70,0 ng/µl
| 1,867
|
| ccdB tet
| 50,0 ng/µl
| 1,818
|
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
| template
| volume
| mass
|
| ccdB amp
| 16µl
| 930ng
|
| ccdB cam
| 14,3µl
| 1µg
|
| ccdB tet
| 16µl
| 800ng
|
| ccdB kan
| 16µl
| 400ng
|
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
| template (~4ng)
| 2µl
|
| Pfu
| 0,5µl
|
| Primer *2
| 2,5µl *2
|
| 10x buffer
| 5µl
|
| dNTP Mix
| 1µl
|
| DMSO
| 1,25µl
|
| H2O
| 35,25µl
|
| sum
| 50µl
|
-> 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:
| PCR nr.
| concentration
| A260/A280
|
| 7b
| 10 ng/µl
| 2,0
|
| 10
| 17,5 ng/µl
| 1,4
|
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; ab: 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:
| PCR nr.
| concentration
| A260/A280
|
| 4aP2
| 12,5 ng/µl
| 1,67
|
| 4bP2
| 72,5 ng/µl
| 1,53
|
New PCR 7a with Pfu and Phusion
template: 190-6, Primer 13,14
Mixture with Pfu
| template (~2ng)
| 1µl
|
| Pfu
| 0,5µl
|
| Primer *2
| 2,5µl *2
|
| 10x buffer
| 5µl
|
| dNTP Mix
| 1µl
|
| DMSO
| 1,25µl
|
| H2O
| 36,25µl
|
| sum
| 50µl
|
-> 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
| PCR4a (~15ng)
| 1.2µl
|
| PCR4b (~15ng)
| 2µl (1:10)
|
| Pfu
| 0.5µl
|
| Primer *2
| 2.5µl *2
|
| 10x buffer
| 5µl
|
| dNTP Mix
| 1µl
|
| DMSO
| 1,25µl
|
| H2O
| 34.05µl
|
| sum
| 50µl
|
-> 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
| template (~2ng)
| 1µl
|
| Pfu
| 0.5µl
|
| Primer *2
| 2.5µl *2
|
| 10x buffer
| 5µl
|
| dNTP Mix
| 1µl
|
| DMSO
| 1,25µl
|
| H2O
| 36.25µl
|
| sum
| 50µl
|
-> 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
| name
| 4a-7
| 4a-8
| 4b-9
| 4b-10
| 3-3
| 3-6
| 6-11
| 6-12
|
| DNA
| 4a; 2.4µl
| 4a; 2.4µl
| 4b; 0.5µl
| 4b; 0.5µl
| 3; 2µl
| 3; 2µl
| 6; 0.5µl
| 6; 0.5µl
|
| primer [1pmol/µl]
| 7; 3.2µl
| 8; 3.2µl
| 9; 3.2µl
| 10; 3.2µl
| 3; 3.2µl
| 6; 3.2µl
| 11; 3.2µl
| 12; 3.2µl
|
| Tris (10mM); pH 8.2
| 1.4µl
| 1.4µl
| 3.3µl
| 3.3µl
| 1.8µl
| 1.8µl
| 3.3µl
| 3.3µl
|
every charge 7µl
New overlapping PCR 5 with Pfu
template: 4a, 4b; 15ng
Primer 7,10
Mixture
| PCR4a (~7ng)
| 0.56µl
|
| PCR4b (~8ng)
| 1.1µl (1:10)
|
| Pfu
| 0.5µl
|
| Primer *2
| 2.5µl *2
|
| 10x buffer
| 5µl
|
| dNTP Mix
| 1µl
|
| DMSO
| 1,25µl
|
| H2O
| 36.6µl
|
| sum
| 50µl
|
-> 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
| template[2ng/µl]
| 1µl
| 2µl
|
| pfu polymerase
| 0.5µl
| 0.5µl
|
| primer13
| 2.5µl
| 2.5µl
|
| primer14
| 2.5µl
| 2.5µl
|
| buffer
| 5µl
| 5µl
|
| dNTP Mix
| 1µl
| 1µl
|
| DMSO
| 1,25µl
| 1.25µl
|
| H2O
| 36.25µl
| 35.25
|
| sum
| 50µl
| 50µl
|
Agarose gel electrophoresis of 7a
-> results: "primer clouds"
charges for sequencing for PCR 1,7b and 9
| name
| 1-1
| 1-2
| 7b-15
| 7b-16
| 9-20
| 9-21
| 10-22
| 10-23
|
| DNA
| 1[1:10]; 1.74µl
| 1[1:10]; 1.74µl
| 7b; 3µl
| 7b; 3µl
| 9; 1.78µl
| 9; 1.78µl
| 10; 3.8µl
| 10; 3.8µl
|
| primer [1pmol/µl]
| 1; 3.2µl
| 2; 3.2µl
| 15; 3.2µl
| 16; 3.2µl
| 20; 3.2µl
| 21; 3.2µl
| 22; 3.2µl
| 23; 3.2µl
|
| Tris (10mM); pH 8.2
| 2.06µl
| 2.06µl
| 0.8µl
| 0.8µl
| 2.02µl
| 2.02µl
| 0µl
| 0µl
|
every charge 7µl
9-07-2010
Agarose gel electrophorese of PCR gel extractions
150V, 25min
-> Protocol: 11 Agarose gel electrophoresis
-result:
from left to right: PCR 1,2a,2b,3,4a,/,7b,9,10,/,ladder,pathway
| PCR nr.
| 1
| 2a
| 2b
| 3
| 4a
| 7b
| 9
| 10
|
| expected band (bp)
| 492
| 332
| 772
| 1087
| 330
| 402
| 808
| 1888
|
| shown band(s)
| 550,200
| 300
| 750
| 1100
| 300
| 450
| 900,1500
| 1900
|
| clean charge
|
| x
| x
| ~x
| x
| x
|
| x
|
new PCR for PCR6
PCR mixture for PCR6
| Concentration
| Low
| High
|
| template
| 1 µl (1:100)
| 15µl (1:100)
|
| primer (11,12)
| 2.5 µl*2
|
|
| dNTPs
| 1 µl
|
|
| Pfu
| 0.5 µl
|
|
| 10xbuffer
| 5 µl
|
|
| DMSO
| 1,25µl
|
|
| H2O
| 36.25 µl
| 22.25 µl
|
| sum
| 50µl
|
|
-> Protocol: Thermal cycler program: Touch down, 62°C-52°C, 30 cycles by 55°C
9-08-2010
Restriction digestion of eGFP, PCR6, ccdBamp
 Gel photo of (left to right) PCR6(5ng/100ng) template as prepatation for ligation
-> Protocol 5 Restriction digestion
| eGFP
|
| SV40PA=PCR6
|
| ccdBamp
|
|
| DNA
| 5µl=300ng
| DNA
| 1µl=37,5ng
| DNA
| 2µl=115ng
|
| Buffer MC
| 2µl
| Buffer D
| 2µl
| Buffer H
| 2µl
|
| BSA 1:10
| 2µl
| BSA 1:10
| 2µl
| BSA 1:10
| 2µl
|
| EcoRI
| 0,5µl
| XbaI
| 0,5µl
| EcoRI
| 0,5µl
|
| SpeI
| 0,5µl
| PstI
| 0,5µl
| PstI
| 0,5µl
|
| H2O
| 13µl
| H2O
| 14µl
| H2O
| 13µl
|
| sum
| 23µl
| sum
| 20µl
| sum
| 20µl
|
1:30h 37°C, 20min 80°C
Agarose gel electrophoresis of PCR6
-> Protocol: 11 Agarose gel electrophoresis
25min, 150V
Ligation 1
| eGFP
| 14,4µL
| (144ng)
|
| SV40PA
| 4,8µL
| (48ng)
|
| ccdBamp
| 2,9µL
| (100ng)
|
| T4 Buffer 10x
| 3µL
|
|
| T4 Ligase
| 0,5µL
|
|
| H2O
| 4,4µL
|
|
| sum
| 30µL
|
|
Ligation at 22,5°C for 30 min, denaturation at 65°C for 10 min.
Concentration of DNA in Ligation 1:
Transformation
-> Protocol 18 competent cells2
The incubation time for the cells is here 1 hour.
9-09-2010
Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9
Agarose gel electrophoresis of PCR 1, 3, 4a, 5, 7a, 9
-> protocol 11 Agarose gel electrophoresis
results: "primer-clouds", PCR 9: no band
Plasmid extraction of ccdBcam, ccdBamp, Bak, CMV, PhiC31o
-> Protocol 4 Plasmid extraction from cells
| Plasmid
| concentration [ng/µL]
| A260/A280
|
| ccdBcam
| 22.5
| 1.0
|
| ccdBamp
| 42.5
| 1.2
|
| Bak
| 17.5
| 0.8
|
| CMV
| 10.0
| 0.7
|
| PhiC310
| 60
| 1.4
|
Eluated with H2O instead of the Eluation Buffer.
New PCR 1, 3, 4a, 5, 7a, 9, 10 with phusion
| PCRnr.
| 1
| 3
| 4a
| 4b
| 5
| 7a
| 9
| 10
|
| template
| pDS7 (1:100); 4µl
| 2a; 0.9µl+2b; 0.6µl
| Bak; 0.5µl
| Bak; 0.5µl
| 4a; 0.8µl+4b; 1µl
| 190-6 (1:100); 1µl
| eGFP (1:25); 2µl
| PhiC31o (1:10); 1µl
|
| primer [10pmol/µl]
| 1,2; 2.5µl
| 3,6; 2.5µl
| 7,8; 2.5µl
| 9,10; 2.5µl
| 7,10; 2.5µl
| 13,14; 2.5µl
| 20,21; 2.5µl
| 22,23; 2.5µl
|
| H2O
| 28µl
| 30.5µl
| 31.5µl
| 31.5µl
| 30.2µl
| 31µl
| 30µl
| 31µl
|
| Annealing temperature
| 51.5°C
| 53.1°C
| 53.1°C
| 51.5°C
| 53.1°C
| 57.5°C
| 61°C
| 57.5°C
|
+ in each assay:
dNTP mix: 1µl, 5x Phusion buffer: 10µl, DMSO:1.5µl, Phusion:0.5µl
sum: 50µl
Program: standard Phusion PCR, 29 cycles; anneling temperatures: see above
-> Protocol: 15 PCR with Phusion
9-10-2010
Agarose gel electrophoresis of PCR 1, 4a, 4b, 5, 7a
gel electrophoresis of PCR 1, 4a, 4b, 5, 7a
Only 4a has been amplified successfully.
Agarose gel extraction of PCR 3, 4a, 5, 9, 10
9-11-2010
weekend
9-12-2010
weekend
9-13-2010
text
9-14-2010
text
9-15-2010
text
9-16-2010
text
9-17-2010
text
9-18-2010
weekend
9-19-2010
weekend
9-20-2010
text
9-21-2010
text
9-22-2010
text
9-23-2010
text
9-24-2010
text
9-25-2010
weekend
9-26-2010
weekend
9-27-2010
text
9-28-2010
text
9-29-2010
text
9-30-2010
text
10-01-2010
text
10-02-2010
weekend
10-03-2010
weekend
|
"
