Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

•  CD79B-RFP-G4 SDM
•  Midiprep and digest
  •  hEF1a:tagBFP
•  Inoculate minis
  •  RFP-IgM CH golden gate
  •  hEF1a:CD79A-linker-G4
•  Miniprep and digest
  •  hEF1a:CD79A-linker-G4
  •  TRE:Syk-15-tTEV
  •  hEF1a:VP16Gal4
  •  EGSH:mKate
  •  hEF1a:VgEcR-2A-RxR
  •  hEF1a:CD79B-TCS-G4
  •  TRE:mKate
•  Inoculate midis
  •  hEF1a:VP16Gal4
  •  EGSH:mKate
  •  hEF1a:VgEcR-2A-RxR
  •  hEF1a:CD79B-TCS-G4
  •  TRE:mKate
•  Sequencing
  •  hEF1a:CD79A-linker-G4
  •  TRE:Syk-15-tTEV
  •  TRE:Syk-RFP-TEV (missing read only)
  •  TRE:Syk-RFP-tTEV (missing read only)

Thursday's To Do's 

•  Innoculate midi cultures
  •  hEF1a:tagBFP (from liquid mini culture)
•  Inoculate mini cultures - because picking from plates of transformed reactions
  •  hEF1a:CD79A-linker-G4 golden gates
  •  TRE:Syk-15-tTEV
  •  TRE:mKate
  •  hEF1a:gal4VP16
  •  EGSH:mKate
  •  hEF1a:VgEcR-2A-RxR
  •  hEF1a:CD79B-TCS-Gal4vp16
•  Transform
  •  RFP-IgM CH golden gate
  •  Failed CD79A linker golden gates
•  Replate failed CD79A linker golden gates
•  Re-golden gate failed golden gates?

Wednesday's To Do's  

•  Transform
  
  •  CD79A linker golden gates
  •  TRE:Syk-15-tTEVp golden gate
    
  •  EGSH:mKate
  •  hEF1a:VgEcR-2A-RxR
  •  hEF1a:gal4vp16
  •  TRE:mKate
  •  hEF1a:CD79B-TCS-Gal4vp16
•  Design additional Syk-TEV Sequencing Primer
•  order primers to re-pcr CD79B-RFP-G4
•  Antibody PCRs
  •  Purify
  •  Gel
  •  Golden Gate
•  Non-Lab To Do's:
  •  Order chicken lysozyme and gp120?
  •  order antibodies for antigen binding detection

:

•  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:

Geneious To Do List:

•  Antibody cloning for chicken lysozyme - Kathryn 
•  hef1a sequencing primersBCR 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  
•  Is gantenerumab proprietary and can we actually publish with it?