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 | |
in midiprep midi culture frozen SEQUENCE after! | pick colonies 8/1617pick colonies | replate 8/ | 1617 | replate 8/1617 | |
6TCS12 | 9TCS12 | 12TCS12 | |||
in midiprep midi culture frozen SEQUENCE after! | midi culture frozen | midi culture frozen | |||
3TCS15 | 9TCS15 | 15TCS15 | |||
seq 8/16 17 (#2) | needs to be retransformed | repeat seq 8/16 17 (#1 & #2) |