Team:Heidelberg/Delftibactin/DelRest
From 2013.igem.org
Del Rest. Creating a 31 kbp plasmid.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
Methods:
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. .
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
Dalalala
test