Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

•  Make Kan X-gal plates
•  Midi:
  •  hEF1a:CymR
  •  hEF1a-CuO:mKate
  •  CMV5-CuO:mKate
  •  TRE:Syk
•  digest, gel:
  •  hEF1a:CymR
  •  hEF1a-CuO:mKate
•  transform:
  •  TRE:Syk-15-tTEVp golden gate
  •  LRs:
    •  EGSH:mKate
    •  hEf1a:VgEcR-2A-RXR
    •  TRE:mKate
    •  hEF1a:Gal4VP16 (remember which pDEST)
    •  hEF1a:CD79B-TCS-Gal4VP16 
    •  hEF1a:tagBFP

Thursday's To Do's

:

•  Midiprep (from freezer)
  •  S9TEV
  •  B15T15
•  Golden Gate
  •  B9T9
  •  B15T9
•  LR
  •  hEF1a-CuO:Gmab H
  •  hEF1a-CuO:Gmab L
   gel verify:
  •  hEF1a:CymR
  •  hEF1a-CuO:mKateCD79A
  •  CMV5hEF1a-CuO:mKate
  •  Syk-15-tTEVp PCR
   Pick colonies for midi
  •  hEF1a:CymR
  •  CD79B
  •  hEF1a-CuO:mKate
  •  CMV5-CuO:mKate
  •  TRE:Syk
•  PCR purify TRE:Syk-15-tTEVp
•  Golden gate TRE:Syk-15-tTEVp
•  LRs:
  •  EGSH:mKate
  •  hEf1a:VgEcR-2A-RXR
  •  TRE:mKate
  •  hEF1a:Gal4VP16 (remember which pDEST)
  •  hEF1a:CD79B-TCS-Gal4VP16 
  •  hEF1a:tagBFP

Wednesday's To Do's

•  Flow cytometry with Brian
•  Order chicken lysozyme and gp120?
•  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?

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

• • 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:
^^^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?