Wednesday's To Do's
- Flow cytometry with Brian
- Analyze flow cytometry data
- Miniprep and verify, then sequence
- CD79A-RFP-G4 SDM
- CD79B-RFP-G4 SDM
- Midiprep (and verify?)
- pEXPR TRE:Syk
- hEF1a:CymR
- hEF1a-CuO:mKate
- CMV5-CuO:mKate
- LRs from Tuesday (that we probably won't have done because LR clonase shortage...)
- PCRs of mRFP and mIgM constant region for hEF1a:mRFP-CH (constant heavy chain)
- Order chicken lysozyme and gp120?
Tuesday's To Do's
- Non-lab to do's
- Design (or find?) sequencing primers for mIgM CH (constant region)
- Plan experiment to test gantenerumab binding to beta-amyloid
- Leader sequences for ordering gBlocks of variable regions?
- Review sequencing
- TRE:Syk-pTet:mRFP-TEV
- TRE:Syk-pTet:mRFP-tTEV
- pENTR CD79B-TCS-G4
- Add dox to cells
- Verify
- hEF1a:eYFP
- TRE:eYFP
- TRE:mKate
- Miniprep and verify
- CD79A-linker-G4 colonies (3T3, 3T9, 3T15)
- CD79A-RFP-G4 SDM colonies
- hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
- Send for sequencing
- CD79A-linker-G4 colonies
- CD79A-RFP-G4 SDM colonies
- Re-transform SDM reactions to hopefully get more colonies to choose from?
- Start midiprep cultures?
- hEF1a:CymR
- hEF1a-CuO:mKate
- CMV5-CuO:mKate
- pEXPR TRE:Syk?
- Pick colonies for miniprep cultures
- pEXPR TRE:Syk?
- CD79A-RFP-G4 SDM
- CD79B-RFP-G4 SDM
- LR - probably can't do today because no LR clonase
- EGSH:mKate
- hEf1a:VgEcR-2A-RXR
- hEF1a:mKate
- hEF1a:Gal4VP16 (remember which pDEST)
- hEF1a:CD79B-TCS-Gal4VP16 (ONLY after sequencing pENTR CD79B-TCS-Gal4VP16)
Monday's To Do's
- make BCR enzyme stock
- digest, gel:
- pENTR mKate
- hEF1a:CD79B-TCS-G4
- hEF1a:Gal4VP16
- sequence:
- TRE:syk-ptet:mrfp-(t)TEVP
- hEF1a:CD79B-TCS-G4
- transform:
- TRE:syk because we don't have a midi
- Re-transform CD79A-TCS-G4 and CD79B-TCS-G4 SDM Golden Gates
- TRE:syk because we don't have a midi
- pick more CD79A linker colonies (all old DNA is not good)
- pick CD79A SDM colony
- pick hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
CMV5-CuO 715:
hEF1a-CuO 715:09
CymR-CuO 710:09
Geneious To Do List:
- 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
- Is gantenerumab proprietary and can we actually publish with it?