Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

:

•  Take out plates
•  Miniprep (if you can find 2mL tubes...)
  •  pENTR HyHEL10 Heavy
  •  hEF1a:CD79B-9T15-G4 
•  Pick colonies
  •  Plated 8/8
    •  A-15T9
    •  TRE:Syk-3-TEVp
    •  TRE:Syk-6-TEVp
    •  TRE:Syk-9-TEVp
    •  TRE:Syk-12-TEVp
    •  B-3T15
    •  B-9T9
    •  TRE:Syk-15-tTEVp
  •  Plated previously
    •  A-3T15
    •  A-9T15
    •  B-15T15
•  Start midiprep cultures
  •  
    B3T3
  •  
    B3T9
  •  B6T6
  •  B9T3
  •  B9T6
  •  B12T6
  •  B15T9

• 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)

Saturday's To Do's

•  Kill LR (TRE:Syk-15-tTEVp)
•  Take out plates + colonies 
•  Transform
  •  hEF1a:CD79A-15T9-G4 (because even though we midi'ed it, sequencing was bad)
  •  TRE:Syk-linker-TEVp (3, 6, 9, 12)
  •  hEF1a:CD79B-3T15-G4
  •  hEF1a:CD79B-9T9-G4
  •  TRE:Syk-15-tTEVp

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 

•  Cytometry time 
•  Order Cumate 
•  Chicken lysozyme, gp120
•  Antibodies for antigen binding detection

Waiting on primers to do list:

• 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?

THINGS TO NOT FORGET TO DO:

• SYK-15tTEVP golden gate when we figure out what's wrong with the AarI
• 3T3 MIDI