Team:Heidelberg/Delftibactin/DelRest
From 2013.igem.org
Del Rest. Creating a 31 kbp plasmid.
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Methods:
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06-07-2013
Amplification from FS_02 to FS_03; 5.3 kb
- Reaction
what | µL |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02: (1/10) | 1 |
FS_03: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1/- |
dd H2O | 6/7 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
63 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- Amplification worked with 5% DMSO
- Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices
03-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
µL 1st PCR | what | µL 2nd PCR |
---|---|---|
1 | D. acidovorans DSM-39 | 1 |
2.5 | FS_02 (1/10) | 2.5 |
2.5 | FS_05 (1/10) | 2.5 |
25 | Phusion Master Mix | 25 |
- | DMSO | 2.5 |
19 | dd H2O | 19 |
- Conditions
Biorad MyCycler | ||
---|---|---|
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 |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- sample has to be purified before further use since contamination with propanol was present
04-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
what | µL 2nd PCR |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02 (1/10) | 2.5 |
FS_05 (1/10) | 2.5 |
Phusion Master Mix | 25 |
DMSO | 2.5 |
dd H2O | 16.5 |
- Conditions
Biorad MyCycler | ||
---|---|---|
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 |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL
- PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp
05-08-2013
Amplification from FS_02 to FS_07; 16.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_02: (1/10) | 1 |
FS_07: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
6 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 5min 40s | |
24 | 98 | 1 |
60 | 5 | |
72 | 5min 40s | |
1 | 72 | 17 min |
1 | 4 | inf |
Results:
- amplification of DelAG did not work
- repeat PCR with higher annealing temperature to increase specifity
06-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
- 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 | 6/7 |
- Conditions
Biorad MyCycler | ||
---|---|---|
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 |
Results:
- Amplification of DelEG worked but another unexpected product was amplified as well
- band was cut out avoiding contamination with other product and DNA purified using QIAquick Gel Extraction Kit
05-07-2013
Amplification from FS_08 to FS_11; 6.5 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_08: (1/10) | 1 |
FS_11: (1/10) | 1 |
Phusion Master Mix | 10 |
dd H2O | 6 |
DMSO | 1 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
14 | 98 | 1 |
62 ↓ 0.5 | 5 | |
72 | 3:20 min | |
16 | 98 | 1 |
60 | 5 | |
72 | 3:20 min | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- A weak band was visible, but it was on the wrong height.
- Accidently the band was cut anyway.
- Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.
07-07-2013
Amplification from FS_08 to FS_11; 6.5 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_08: (1/10) | 2.5 |
FS_11: (1/10) | 2.5 |
Phusion Master Mix | 25 |
dd H2O | 19 |
DMSO | 2.5 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 2:30 min | |
18 | 98 | 1 |
63 | 5 | |
72 | 2:30 min | |
1 | 72 | 10 min |
1 | 4 | inf |
Results:
- There was no band visible on the gel.
- Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.
04-07-2013
Amplification from FS_12 to FS_13; 2.7 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_12: (1/10) | 1 |
FS_13: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
72 | 40 | |
1 | 72 | 420 |
1 | 4 | inf |
Results:
- Amplification of DelOP did not work
- Amplification will be repeated to exclude that pipetting errors were the reason for the failure
04-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_14: (1/10) | 1 |
FS_15: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1 |
dd H2O | 6 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles | temperature [°C] | Time [s] |
1 | 98 | 10 |
30 | 98 | 1 |
72 | 40 | |
1 | 72 | 7 min |
1 | 4 | inf |
Results:
- Amplification worked very well, we had a bright band on the right height.
- Band was cut out and DNA purified using QIAquick Gel Extration Kit.
06-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
what | µl |
---|---|
D. acidovorans DSM-39 | 1 |
FS_14: (1/10) | 1 |
FS_15: (1/10) | 1 |
Phusion Master Mix | 10 |
DMSO | 1/- |
dd H2O | 6/7 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 10 |
12 | 98 | 1 |
66 ↓ 0.5 | 5 | |
72 | 42 | |
18 | 98 | 1 |
63 | 5 | |
72 | 42 | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
05-07-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
what | µl |
---|---|
Template pSB4K5 | 1 |
FS_16: (1/10) | 1 |
FS_01: (1/10) | 1 |
Phusion Master Mix | 10 |
dd H2O | 6 |
DMSO | 1 |
- Conditions
Biorad MyCycler | ||
---|---|---|
Cycles-PCR | temperature [°C] | Time [s] |
1 | 98 | 5 |
14 | 98 | 1 |
62 ↓ 0.5 | 5 | |
72 | 3:20 min | |
16 | 98 | 1 |
60 | 5 | |
72 | 3:20 min | |
1 | 72 | 12 min |
1 | 4 | inf |
Results:
- Amplification worked well and band was at expected height. Nevertheless the gel band did not run properly on the gel.