Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 Monday

 To do's

:

•  Midiprep
  •  something
  (from freezer)
  •  S9TEV
  •  B15T15
•  Golden gates
  •  hEF1a:CD79B-pTet:RFP-Gal4VP16
  •  pENTR Syk-15-tTEV
  •  pENTR VRC01 light
  •  pENTR VRC01 heavy
  •  pENTR HyHEL-10 light
  •  pENTR HyHEL-10 heavy
•  Pick Colonies for mini
  •  TRE:Syk-3-tTEV
  •  TRE:Syk-6-tTEV
  •  TRE:Syk-9-tTEV
  •  TRE:Syk-12-tTEV
  •  hEF1a:CD79A-9T15-G4
•  Inoculate midis
  •  From failed midiprep
    •  hEF1a:Gal4
    •  GGDonr
    •  Linkers
  •  Additional linkers
    •  CD79A-6T9
    •  CD79A-9T12

Tuesday's to Dos

•  Transform golden gates
  •  hEF1a:CD79B-pTet:RFP-Gal4VP16
  •  pENTR Syk-15-tTEV
  •  pENTR VRC01 light
  •  pENTR VRC01 heavy
  •  pENTR HyHEL-10 light
  •  pENTR HyHEL-10 heavy
•  Miniprep and digest
  •  TRE:Syk-3-tTEV
  •  TRE:Syk-6-tTEV
  •  TRE:Syk-9-tTEV
  •  TRE:Syk-12-tTEV
  •  hEF1a:CD79A-9T15-G4
•  Midiprep and digest
  •  From yesterday's failed midipreps
    •  hEF1a:Gal4
    •  GGDonr
    •  Linkers
  •  Additional linkers
    •  CD79A-6T9
    •  CD79A-9T12
•  Pick colonies for failed linkers
  •  CD79A-3T15
  •  CD79A-12T9
  •  CD79A-15T9
•  Send for sequencing
  •  Resend 12T12 (1), also send 12T12 (2)
  •  9T15 (1) and (2)

Tuesday's to Dos

•  Transform golden gates
  •  hEF1a:CD79B-pTet:RFP-Gal4VP16
  •  pENTR Syk-15-tTEV
  •  pENTR VRC01 light
  •  pENTR VRC01 heavy
  •  pENTR HyHEL-10 light
  •  pENTR HyHEL-10 heavy
•  Miniprep and digest
  •  TRE:Syk-3-tTEV
  •  TRE:Syk-6-tTEV
  •  TRE:Syk-9-tTEV
  •  TRE:Syk-12-tTEV
  •  hEF1a:CD79A-9T15-G4
•  Midiprep and digest
  •  From yesterday's failed midipreps
    •  hEF1a:Gal4
    •  GGDonr
    •  Linkers
  •  Additional linkers
    •  CD79A-6T9
    •  CD79A-9T12
•  Inoculate midiprep cultures for successful CD79A linkers (based on sequencing data that we should get back today) and pick new colonies for not successful ones

• 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:
^^^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