Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Monday's To Do's 

•  make BCR enzyme stock
•  digest, gel:
  •  pENTR mKate
  •  hEF1a:CD79B-TCS-G4
  •  hEF1a:Gal4VP16
•  LR EGSH:mKate, hEf1a:VgEcR-2A-RXR, hEF1a:mKate
•  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 
•  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

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