PCR
| Construct | F Tm | R Tm | Rxn T | Expected band size |
|---|---|---|---|---|
| CD79A | 61 | 60 | 63 | 750 bp |
| CD79B | 66 | 67 | 69 | 750 bp |
| Gal4VP16 | 66 | 62 | 65 | 750 bp |
| pTEt:mRFP | 51 | 58 | 61 | 800 bp |
03/31 and 04/01
Construct | F Tm | R Tm | Rxn T |
|---|---|---|---|
| CD79A | 61 | 60 | 65 |
| CD79B | 66 | 67 | 71 |
| Gal4VP16 | 66 | 62 | 65 |
| pTEt:mRFP | 51 | 58 | 61 |
04/10
Construct | F Tm | R Tm | Rxn T |
|---|---|---|---|
| CD79A | 61 | 60 | 65 |
| CD79B | 66 | 67 | 71 |
| Gal4VP16 | 66 | 62 | 65 |
| pTEt:mRFP | 51 | 58 | 61 |
Construct | F Tm | R Tm | Rxn T |
|---|---|---|---|
| CD79A | 69.2 | 70.7 | 70 |
| CD79B | 69.1 | 73 | 71 |
AarI Golden Gate
Gel extraction
AarI golden gate
transformation of golden gate
05/04
pick colonies
miniprep and restriction digest verification
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SDM
Internal BsaI cut site in Gal4VP16 is messing with our golden gates --> SDM BsaI site
golden gate based SDM using EspI
PCR purification
DpnI digest
transformation - no colonies
07/05
replate remainder of 07/02 transformation
retransform SDM 10uL DNA - plate all of it
07/06
pick colonies
07/07
send for sequencing - no priming --> repeat read #4 (gal4)
07/09
CD79A #1 read 4 (gal4) shows correct mutation --> repeat sequencing for remainder of construct
07/14
Remainder of sequencing shows correct sequence for CD79A and pTet:mRFP - construct ready for BsaI golden
03/23 PCRs
 | Ladder; CD79A; CD79B; pTet:mRFP | Notes: CD79A and CD79B bands are not of the correct size. They appear to have primer dimers. Temperature used for the PCR may not be appropriate, since we changed PCR reagents from Phusion to Q5. Gal4VP16 and pTet:mRFP construct might be correct - too much DNA in the lane to discern exact position of the band. PCRs will be redone with correct temperatures and less template. |
03/31 and 04/01 PCRS
 |  | Notes:(Lanes 1-5 had holes in the bottom, had to load additional lanes because first samples were lost - no additional DNA from PCR reaction) Gal4VP16 and pTet:mRFP bands are the correct size CD79A and CD79B constructs show bands of smaller size. Since we're redoing the Gal4VP16 and pTet:mRFP PCRs anyway we can redo the CD79 PCRs but increase the annealing temperature by 2C |
..but we have to redo the PCR reaction because we don't have any PCR product left. 04/10 PCRs
|  | Notes:Gal4VP16 and pTet:mRFP constructs have the correct band size --> ready for AarI golden gate CD79A and CD79B PCRs still seem to have primer dimers.CD79A/B primers have to be redesigned. |
04/25 PCRs
|  | Notes:CD79A construct have the correct band size --> ready for AarI golden gate CD79B PCRs still seem to have primer dimers. CD79B primers have to be redesigned. |
05/06 Restriction verification
Enzyme used: BamH1
Expected | Ladder | Gel | Notes |
|---|---|---|---|
 |  |  | Lanes are Ladder, hEF1a:CD79A-pTet:mRFP-Gal4VP16 sample 1, 2, 3, 4, 5. Only samples 1 and 5 appear to have succeeded.
^ that makes sense because only samples 1 and 5 had (very) red bacteria when I spun them down for the miniprep (even though I picked 5 red colonies):
It's interesting that we don't see the lower size bands (i guess that could be because the gel is very jank towards the end) |
Sequencing
