Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's:

•  pour kan x-gal plates 
•  restreak TRE:Syk-mRFP-(t)TEVp
•  pick TRE:Syk-mRFP-(t)TEVp
•  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 
•  pLac:mKate pick colonies
•  ScaI to lab stock

Geneious To Do List:

•  

  SDM primers: CD79B, Gal4VP16, TEVp

•  TRE:gal4vp16 sequencing primers
•  TRE:Syk-mRFP-(t)TEVp sequening primers
•  hef1a sequencing primers

•  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)

Sequencing account information:BCR 2.0 To Do
^^^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?