hEF1a:CD79B-pTet:mRFP-Gal4VP16
PCR: when we redesigned primers to fix dimerization issues, we accidentally introduced a missense mutation in the third codon from the end of CD79B. We decided to use SDM to fix the mutation instead of rePCRing
- SDM
- Gel verification of SDM --> no bands: possible that there wasn't enough amplification. will run SDM again and transform product directly
- SDM and DpnI digest
- transform
- No colonies --> could be because a small amount of the digest was transformed.
- all of digest transformed
- No colonies
hEF1a:CD79B-XnTCSXc-Gal4VP16
| 3TCS3 | 9TCS3 | 15TCS3 | ||||
| midi culture frozen | ||||||
| 6TCS6 | 9TCS6 | 12TCS6 | ||||
| 3TCS9 | 6TCS9 | 9TCS9 | 12TCS9 | 15TCS9 | ||
| seq 8/13 |
| seq #2 8/12 | repeat seq. w/ alt. prot. (8/11) | 26 |
| plated 8/26in mini culture 8/12 |
| 6TCS12 | 9TCS12 | 12TCS12 | ||||
| seq 8/12 (#2) | midi culture frozen | midi culture frozen
|
| |||
| 3TCS15 | 9TCS15 | 15TCS15 | ||||
| in seq 8/10 | transformed 8/12 | in seq |