BCR 2.0

Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Tuesday's To Do's

• LR
  • EGSH:mKate
  • hEf1a:VgEcR-2A-RXR
  • hEF1a:mKate
  • hEF1a:Gal4VP16 (remember which pDEST)
  • hEF1a:CD79B-TCS-Gal4VP16 (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 
• 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