Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

:

•  Flow cytometry with Brian
•  Midiprep (and verify?)
  •  pEXPR TRE:Syk
  •  hEF1a:CymR
  from freezer)
  •  S9TEV
  •  B15T15
•  Golden Gate
  •  B9T9
  •  B15T9
•  LR
  •  hEF1a-CuO:mKateGmab H
  •  CMV5hEF1a-CuO:mKate
•  PCRs of mRFP and mIgM constant region for hEF1a:mRFP-CH (constant heavy chain)
•  Order chicken lysozyme and gp120?
•  LR - probably can't do today because no LR clonase
  •  EGSH:mKate
  •  hEf1a:VgEcR-2A-RXR
  •  hEF1a:mKate
  •  hEF1a:Gal4VP16 (remember which pDEST)
  •  hEF1a:CD79B-TCS-Gal4VP16 

Non-lab to do's

•  
  • •  Design (or find?) sequencing primers for mIgM CH (constant region)
    •  Plan experiment to test gantenerumab binding to beta-amyloid
    •  Leader sequences for ordering gBlocks of variable regions?
    •  Review sequencing
      •  TRE:Syk-pTet:mRFP-TEV - priming issue
      •  TRE:Syk-pTet:mRFP-tTEV - priming issue
      •  pENTR CD79B-TCS-G4 - correct

CMV5-CuO 715:

hEF1a-CuO 715:09

CymR-CuO 710:09

Geneious To Do List:

•  Antibody cloning for chicken lysozyme - Kathryn 
•  hef1a sequencing primers

• • 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?