Friday's To Do's
- midi pLac:mKate
- mini pENTR hEF1a-LacO, pENTR LacI
- mini culture pENTR mKate
- mini pENTR mKate
digest all midis
digest CD79A-linker-G4
- verification digests, gels
LR EGSH:mKate, hEf1a:VgEcR-2A-RXR
restreak TRE:Syk-pTET:mRFP-(t)TEVp
sequence TRE:syk-ptet:mrfp-(t)TEVP
TRE:Syk mini transformation
MAV1212 transformation
Thursday's To Do's
- Kill LRs
- DpnI digest SDM
- SDM CD79B-RFP-G4 PCR purification
- Anneal Syk Linker oligos
- Transform:
- hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate LRs
- SDM CD79B-RFP-G4
- pENTR mKate
- pick pENTR LacI, pENTR hEF1a-LacO colonies
- midi TRE:Gal4VP16, TRE:mKate, hEF1a:LacI
- digest, gel pLac:mKate LR #2
- grow up pLac:mKate midi culture
Wednesday's To Do's
- SDM cd79b-ptet:mrfp-gal4vp16 - Christian
- Innoculate Midi TRE:gal4VP16 (2nd transformation #1) - Lyla
- Transform:
- pENTR LacI and pENTR pLac - Lyla
- Miniprep, digest, gel
- TRE:Syk-pTet:mRFP-TEVp
- pLac:mKate
- TRE:gal4vp16 #2 (should be in unverified mini/midi in 4C) - see cloning progress 06/26
- rerun digest/gel from yesterday (TRE:mKate and hEF1a:LacI LR1 #2,3 with SalI and ApaI resp.)
- LR hef1a:cymR and hEF1aCuO:mkate (look to see if CMV5CuO is in lab promoter box - report number to Brian) - Lyla
CMV5-CuO 715:
hEF1a-CuO 715:09
CymR-CuO 710:09
Geneious To Do List:
-
SDM primers: CD79B, Gal4VP16, TEVp
- TRE:gal4vp16 sequencing primers
- TRE:Syk-mRFP-(t)TEVp sequening primers - Kathryn
- Antibody cloning for chicken lysozyme - Kathryn
-
hef1a sequencing primers
^^^Email: bcrpeeps@mit.edu
^^^Pass: grapefruit
Higher Level To Do's:
- Decide on other antigen-variable regions we want to test
- Other experimental planning (on experimental page)
- Parts list (excel sheet detailing specs/locations of parts)
- How do we do site directed mutagenesis?
- Literature research:
- kozak sequences for heavy and light chains and CD79s
- truncated TEVp
- other antigens to detect using BCR (we were thinking of small molecule detection)
- inducible promoters (we were considering LacI - an inducible repressor - but we want to look into other options first)
- Cloning planning
- hEF1a:Syk-pTet:mRFP-TEVp
- variable region