BCR 2.0

Cloning Strategy

Linker Optimization

Antibody Choices 

Today's To Do's:

• restreak TRE:Syk-mRFP-(t)TEVp - eric 
• miniprep digest verify TRE:Syk-mRFP-(t)TEVp
• transform pENTR LacI and pENTR pLac
• pick hEF1a:LacI, TRE:mKate, pLac:mKate
• digest, gel hEF1a:LacI, TRE:mKate, pLac:mKate LR2
• miniprep TRE:gal4 transformation 2 
• digest, gel verify TRE gal4 transformation 2
• split cells - Lyla 
• pLac:mKate pick LR colonies - Lyla 
• LR hef1a:cymR and cmv5CuO:mkate
• ScaI to lab stock

Wednesday's To Do's

• Transform: 
  • LR hef1a:cymR and cmv5CuO:mkate
  • pENTR LacI and pENTR pLac
• Miniprep, digest, gel 
  • TRE:Syk-pTet:mRFP-TEVp
  • pLac:mKate

Geneious To Do List:

• SDM primers: CD79B, Gal4VP16, TEVp

• TRE:gal4vp16 sequencing primers
• TRE:Syk-mRFP-(t)TEVp sequening primers - 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