BCR 2.0

Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

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
• LR pENTR EGSH, pENTR VgEcR-2A-RXR
• midi TRE:Gal4VP16, TRE:mKate, hEF1a:LacI
• restreak TRE:Syk-pTET:mRFP-(t)TEVp

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 

Tomorrow 

• transform pENTR mKate

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

BCR 2.0 To Do

^^^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