Cloning Strategy

Linker Optimization

Antibody Choices 

Today's To Do's:

• syk-rfp-tvep/ttvep linker golden gates (cidar and overnight)
• gel verify TRE:Gal4VP16 digest
• order TRE:gal4vp16 sequencing primers 
• send TRE:gal4vp16 for sequencing
• miniprep hEF1a:LacI, TRE:mKate, pLac:mKate colonies 
• transform LRs
• transform golden gates
• transform pENTR LacI and pENTR pLac
• order new SDM primers 
• send TRE:Gal4VP16 for sequencing 

Other Immediate To Do's:

• Innoculate TRE:mKate midi culture (culture number 2)
• transform golden gates
• get cells from Jeremy

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