Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

•  Miniprep, digest, gel, sequence:
  •  hEF1a:CD79B-pTET:mRFP-G4
  •  A-15T9
  •  A-9T15
  •  A-3T15
  •  T:S-12-tT both cultures look red 
  •  T:S-3-tT one of the cultures looks okay (not red)
•  Innoculate midi
  •  A-12T12
•  LR
  •  Syk-15-tTEVp pENTR with TRE
•  Redigest midis, gel 
  •  A-3T3 midi
  •  A-12T9 midi
•  Re-transform (double-check this before re-transforming all of them)
  •  TRE:S-3-tT
  •  TRE:S-6-tT 
  •  TRE:S-9-tT 
  •  TRE:S-12-tT

Friday's To Dos

•  Minipreps
•  Midi A-12T9
•  Digest/gel:
  •  hEF1a:Gal4 + undigested control 
  •  HyHel10 
  •  CD79A-3T3-G4
  •  Minis
  •  Midi
•  Split cells 
•  Sequence constructs w/ correct gels
•  Analyze sequencing 
•  Innoculate midis for constructs w/ correct sequencing 
•  Pick colonies from replated GG transformations 

Thursday's To Dos

•  Transform golden gates
•  Minipreps - linkers
•  Midipreps - ggdonr and a-3t3
•  Digest/gel
•  Inoculate midiprep of hEF1a:CD79A-12T9-G4
•  Pick colonies
  •  Transformed golden gates
    •  hEF1a:CD79B-RFP-G4
    •  pENTR HyHEL-10 light
    •  pENTR Syk-15-tTEV
    •  Syk linkers (3, 6, 9, 12)
  •  hEF1a:CD79A-12T12-G4
  •  hEF1a:CD79A-9T15-G4
•  Split cells

Wednesday's to Dos

•  Redigest basically everything from yesterday and gel
•  Pick colonies
  •  TRE:Syk-15-tTEV
  •  hEF1a:CD79B-RFP-G4
  •  pENTR VRC01 heavy
  •  pENTR VRC01 light
  •  pENTR HyHEL-10 heavy
  •  pENTR HyHEL-10 light
•  Fix - see here for ideas
  •  TRE:Syk-15-tTEV
  •  hEF1a:CD79B-RFP-G4
  •  Syk linkers
•  Miniprep andn digest
  •  CD79A-3T15
  •  CD79A-12T9
  •  CD79A-15T9
•  Inoculate midipreps if the sequencing comes back good
•  Regolden gate things - extra ligation for BsaI golden gate

:

•  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:
^^^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