From 2008.igem.org
(Difference between revisions)
|
|
Line 21: |
Line 21: |
| program:<br> | | program:<br> |
| | | |
- | 1. 95°C - 3' | + | 1. 95°C - 3'<br> |
- | 2. 95°C - 30" | + | 2. 95°C - 30"<br> |
- | 3. 55°C - 45" | + | 3. 55°C - 45"<br> |
- | 4. 68°C - 1' | + | 4. 68°C - 1'<br> |
- | 5. go to step 2 25 x | + | 5. go to step 2 25 x<br> |
- | 6. 68° - 10' | + | 6. 68° - 10'<br> |
- | 7. keep in 4° | + | 7. keep in 4°<br> |
| | | |
| gel electrophoresis<br> | | gel electrophoresis<br> |
Revision as of 16:49, 26 September 2008
|
|
|
|
Preparation of constructs with OmpA protein fusions and Cloning of protein Z DNA to OmpA constructs
1. Isolation of plasmids from cultures inocluated on previous day (pCACYC177 + OmpA_alpha and pACYC177+OmpA_Z_omega).
2. Control digestation of isolated plasmids with BamHI and SacI (we found good clones for both ligations).
Linked PCR Omega-A
reisolated PCR product PCR-omega - 4 µl
reisolated PCRT product A-homo - 13.5 µl
primer OmegaLS - 2 µl
primer APNot - 2 µl
Pfu buffer with Mg2+ - 5 µl
dNTPs - 1 µl
H2o - 22 µl
program:
1. 95°C - 3'
2. 95°C - 30"
3. 55°C - 45"
4. 68°C - 1'
5. go to step 2 25 x
6. 68° - 10'
7. keep in 4°
gel electrophoresis
|
|