Team:Heidelberg/Templates/Del week2 EG
From 2013.igem.org
Contents |
09-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
File:20130709 DelOP.png
PCR for Amplification of DelEG (09.07), lane1=Marker, lane2 DelG=(touchdown), Lane3= DelEG (constant annealing temperature)(11kbp); run at 100 V, 0.8 % gel (TAE),
Wrong description (actually OP) -->picture in wrong section
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 1 |
FS_07: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions I
Cycles-PCR | temperature [°C] | Time [s] |
---|---|---|
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 3 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3 min | |
1 | 72 | 10 min |
1 | 4 | inf |
- Conditions II
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
5 | 98 | 1 |
68 | 5 | |
72 | 3 min | |
25 | 98 | 1 |
72 | 5 | |
72 | 3 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification of DelEG did not work
11-07-2013
Amplification from FS_04 to FS_11s; 17.5 kb
File:20130712 DelLP DelFG DelEG DelEG(PHII).png
PCR for amplification of DelLP, DelFG and DelEG; lane1=Ladder log2, lane2=delLP(11.07), lane3=DelFG(11.07), lane4=DelEG (Phusion flash, 11.07), lane5=DelEG (Phusion II,11.07), lane6=1kb ladder plus; DelLP=6.4kbp, DelFG=5.3kbp, DelEG=16.4kbp; run at 100 V, 0.8 % gel (TAE)
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 1 |
FS_11_short: (1/10) | 1 |
Phusion flash Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 5:30 | |
18 | 98 | 1 |
66 | 5 | |
72 | 5:30 | |
1 | 72 | 15min |
1 | 12 | inf |
Results:
- Amplification of DelEG did not work
- Experiment will be repeated with NEB Phusion II Polymerase as Phusion II is not provided as mastermix and therefore GC-buffer can be used
Amplification from FS_04 to FS_11; 17.5 kb; Phusion II
File:20130712 DelLP DelFG DelEG DelEG(PHII).png
PCR for amplification of DelLP, DelFG and DelEG; lane1=Ladder log2, lane2=delLP(11.07), lane3=DelFG(11.07), lane4=DelEG (Phusion flash, 11.07), lane5=DelEG (Phusion II,11.07), lane6=1kb ladder plus; DelLP=6.4kbp, DelFG=5.3kbp, DelEG=16.4kbp; run at 100 V, 0.8 % gel (TAE)
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 1 |
FS_11_short: (1/10) | 1 |
Phusion II | 0.2 |
DNTP | 0.4 |
Buffer | 4 |
DMSO | 0.6 |
dd H2O | 11.8 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 30 |
12 | 98 | 5 |
68 ↓ 0.5 | 30 | |
72 | 8:10 | |
18 | 98 | 5 |
66 | 30 | |
72 | 8:10 | |
1 | 72 | 15min |
1 | 17 | inf |
Results:
- Amplification of DelEG did not work
- it seems not to be possible to amplify the desired 17 kbp fragent with the chosen primers and the given template, primercombination will be changed in further amplification attempts
14-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
File:20130714 FS04 TO FS07.png
PCR for amplification of DelEG (FS04-FS07, 14.07); lane1=log2 Marker, lane2=DelEG (protocol 68touchdown), lane3=DelEG (protocol 72touchdown); desired amplicon size=11kbp run at 100 V, 0.8 % gel (TAE)
File:20130714 FS04 TO FS07 cut.png
PCR for amplification of DelEG (FS04-FS07, 14.07) after excision; run at 100 V, 0.8 % gel (TAE)
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_04: (1/10) | 2 |
FS_07: (1/10) | 2 |
Phusion flash Master Mix | 10 |
DMSO | 1 |
dd H2O | 4 |
- Conditions I
Cycler incubation room right
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
68 ↓ 0.5 | 5 | |
72 | 3:00 min | |
18 | 98 | 1 |
66 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 12 | inf |
- Conditions II
Cycler incubation room left
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
70 ↓ 0.5 | 5 | |
72 | 3:00 min | |
18 | 98 | 1 |
68 | 5 | |
72 | 3:00 min | |
1 | 72 | 10 min |
1 | 12 | inf |
Results:
- Amplification of DelEG worked with a touchdown PCR starting from 70°C annealing temperature
- band was cut out and DNA purified using QIAquick Gel Extraction Kit
- PCR will be repeated to increase the amount of DNA and gather the concentrations necessary for Gibson Assembly