Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 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 

:

•  Midiprep (from freezer)
  •  S9TEV
  •  B15T15
•  Golden Gate
  •  B9T9
  •  B15T9
•  LR
  •  hEF1a-CuO:Gmab H
  •  hEF1a-CuO:Gmab L
  •  hEF1a-CuO:CD79A
  •  hEF1a-CuO:CD79B
  •  hEF1a-CuO:Lyn
•  Digest all of the Syk-TEVp and Syk-tTEVp midis just to be sure (since there's an internal cut site that didn't get read in sequencing)

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

Sequencing account information:

^^^Email: bcrpeeps@mit.edu

^^^Pass: grapefruit

## Digest tTEVp and TEVp midis just to be sure (since there's an internal cut site)

Non-Lab To Do's 

•  Bug Brian about ordering antibody testing supplies

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 
• Is gantenerumab proprietary and can we actually publish with it?