| Date | Summary |
|---|
| 07/01 | - hEF1a:CD79B-pTET:mRFP-Gal4VP16 CD79B mutation
- hEF1a:CD79A-pTET:mRFP-Gal4VP16 Gal4VP16 mutation
- pLac:mKate miniprep, digest, gel
- TRE:syk-pTet:mRFP-(t)TEVp miniprep, digest, gel
- hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate LR
- pENTR LacI, pENTR hEF1a-LacO transformations
- TRE:Gal4VP16, TRE:mKate, hEF1a:LacI midi culture
|
07/02 | Cumate Switch: - Kill LR
- Transform LR product
Lac Switch: - digest, gel verify hEF1a-LacO:mKate LR#2
- midi culture hEF1a-LacO:mKate LR#2 Sample 2
SDM: - PCR purify CD79B QuickChange SDM
- DpnI digest:
- transform
MIDIs: - TRE:Gal4VP16
- TRE:mKate
- hEF1a:LacI
Mini Cultures: - pENTR hEF2a-LacO
- pENTR LacI
|
| 07/03 | - Verification digests of all midis + redigests of CD79A linker golden gates
- midi pLac:mKate
- mini pENTR hEF1a-LacO and pENTR LacI + digest
- pick pENTR mKate + mini
SDM plates didn't grow any colonies |
| 07/05 | - Transform TRE:Syk-pTet:RFP-TEV, TRE:Syk-pTet:RFP-tTEV, hEF1a:CD79A-pTet:RFP-gal4VP16 SDM, hEF1a:CD79B-pTet:RFP-Gal4VP16 SDM
- Plate remainder of failed SDMs
|
| 07/06 | - digest, gel:
- pENTR mKate
- hEF1a:Gal4VP16
- pENTR CD79B-TCS-Gal4VP16
- sequence:
- pENTR CD79B-TCS-Gal4VP16
- TRE:syk-pTET:mRFP-(t)TEVp
- transform:
- TRE:syk
- Re-transform CD79A-TCS-G4 and CD79B-TCS-G4 SDM Golden Gates
- pick colonies:
- CD79A-3TCSX-Gal4VP16 colonies
- CD79A SDM colonies
- hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
|
| 07/07 | - minprep:
- CD79A-linker-Gal4VP16 (3T3, 3T9, 3T15)
- CD79A-pTet:mRFP-Gal4VP16 Gal4 Bsa1 SDM
- hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
- digest, gel:
- hEF1a:eYFP
- TRE:eYFP
- TRE:mKate
- CD79A-linker-Gal4VP16 (3T3, 3T9, 3T15)
- CD79A-pTet:mRFP-Gal4VP16 Gal4 Bsa1 SDM
|
| 07/08 | - digest hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
- TRE:Syk-15-tTEVp PCR
- Analyze sequencing:
- TRE:Syk-pTet:mRFP-TEVp - priming issue
- TRE:Syk-pTet:mRFP-tTEVp - priming issue
- pENTR CD79B-TCS-G4 - correct
|
| 07/09 | - gel:
- TRE:Syk-15-tTEVp PCR purification
- TRE:Syk-15-tTEVp golden gate
- LRs:
- EGSH:mKate
- hEf1a:VgEcR-2A-RXR
- hEF1a:Gal4VP16
- TRE:mKate
- hEF1a:CD79B-TCS-Gal4VP16
- hEF1a:tagBFP
- Midi Cultures
|
| 07/10 | - Repeat sequencing of CD79A Gal4 SDM #1 since the gal4 read (#4) showed correct mutaiton
- midi cultures
- hEF1a:CymR
- hEF1a-CuO:mKate
- CMV5-CuO:mKate
- TRE:Syk
- Transformations:
- RE:Syk-15-tTEVp golden gate
- Golden gate donor
- LRs:
- EGSH:mKate
- hEf1a:VgEcR-2A-RXR
- TRE:mKate
- hEF1a:Gal4VP16 (remember which pDEST)
- hEF1a:CD79B-TCS-Gal4VP16
- hEF1a:tagBFP
|
| 07/11 | |
| 07/12 | |
| 07/13 | - Reinnoculate midis:
- Pick colonies:
- EGSH:mKate
- hEf1a:VgEcR-2A-RXR
- TRE:mKate
- hEF1a:Gal4VP16 (remember which pDEST)
- hEF1a:CD79B-TCS-Gal4VP16
- hEF1a:tagBFP
|
| 07/14 | - Midis:
- Minipreps, digest, gel:
- cultures didn't grow: hEF1a:gal4, hef1a:VgECr, hef1a: cd79B-TCS-G4, EGSH:mKate --> reinnoculated
- Golden gates:
- cd79A-linker-g4
- TRE:syk-15-ttevp
|
| 07/15 | - IgM constant region + RFP PCRs +gel
- IgM-RFP golden gate
- Transform CD79A linker golden gates and remainder of LRs for cultures that didn't grow today:
- TRE:Syk-15-tTEVp
- TRE:mKate
- EGSH:mKate
- hEF1a:VgEcR-2A-RxR
- hEF1a:Gal4VP16
- hEF1a:CD79B-TCS-Gal4VP16
- Ordered Primers:
- TRE:Syk-pTet:mRFP-TEVp sequencing primers
- hEF1a:Cd79B-pTet:mRFP-Gal4VP16 PCR primers
|
| 07/16 | |
| 07/17 | |