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Team:Heidelberg/Tyrocidine week19 biobrickmod
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== Tyrocidine-Indigoidine-Fusion - Standardization of Constructs == | == Tyrocidine-Indigoidine-Fusion - Standardization of Constructs == | ||
So far, the Tyrocidine-Indigoidine-fusion constructs had two illegal cutting sites of RFC-10 restriction enzymes. Hence, we tried a CPEC-approach for which we reamplified a fragment from the vector with primers that introduced a mutation at the desired position. | So far, the Tyrocidine-Indigoidine-fusion constructs had two illegal cutting sites of RFC-10 restriction enzymes. Hence, we tried a CPEC-approach for which we reamplified a fragment from the vector with primers that introduced a mutation at the desired position. | ||
Revision as of 21:51, 2 October 2013
Contents |
Tyrocidine-Indigoidine-Fusion - Standardization of Constructs
So far, the Tyrocidine-Indigoidine-fusion constructs had two illegal cutting sites of RFC-10 restriction enzymes. Hence, we tried a CPEC-approach for which we reamplified a fragment from the vector with primers that introduced a mutation at the desired position.
Amplification
| what | µl |
|---|---|
| pPW05 (dil.) | 1 |
| RB68 | 2 |
| RB69 | 2 |
| Phusion Flash 2x Master Mix | 10 |
| ddH20 | 5 |
| Cycles | temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 0:05 |
| 35 | 98 | 0:05 |
| 59 | 0:10 | |
| 72 | 3:00 | |
| 1 | 72 | 10:00 |
| 1 | 10 | inf |
Results
File:PCR 1' 2' 3'.png
100px
CPEC
After gel-extraction of the large fragment, the following procedure was followed:
- 22µl H2O, 20µl of the large fragment, 2µl of the short fragment, 1µl DpnI and 5µl 10x CutSmart buffer were incubated for 2 hours at 37°C
- Mixture was purified with isoprop, washed with ethanol and eluted in 10µl H2O
- 10µl Phuson Flash Master Mix was added and the followin protocol was run:
| Cycles | temperature [°C] | Time [min:s] |
|---|---|---|
| 1 | 98 | 0:10 |
| 5 | 98 | 0:01 |
| 53 | 0:05 | |
| 72 | 3:00 | |
| 1 | 72 | 10:00 |
| 1 | 10 | inf |
Unfortunately the efficiency of this was not high enough, as no colonies were visible on the plates after heat-shock transformation in BAP-I cells. The Amplification and CPEC will be repeated next week.
