Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

:

•  Mini, digest
  
  •  TRE:Syk-15-tTEVp

•  Mini, sequence
  
  •  B-3T3-G4
  •  B-3T9-G4
  •  B-6T6-G4
  •  B-6T9-G4
  •  B-9T3-G4
  •  B-9T6-G4
  •  B-9T12-G4
  •  B-12T6-G4
  •  B-12T9-G4
  •  B-12T12-G4
  •  B-15T3-G4
  •  B-15T15-G4

•  Resend for sequencing
  
  •  A-3T15-G4
  •  A-9T15-G4
  •  TRE:Syk-9-tTEVp
  •  TRE:Syk-12-tTEVp

•  Golden Gate, 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
  •  (also transform TRE:Syk-pET:mRFP-TEVp because we don't have enough of it)

•  Streak onto new plate (some white colonies hidden with red ones)
  
  •  hEF1a:CD79B-6T12-G4
  •  hEF1a:CD79B-15T9-G4

•  Pick colonies
  •  pENTR HyHEL10 Heavy
  •  hEF1a:CD79B-9T15-G4

•  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)

Thursday's To Do's 

•  Mini, Midi, digest, gel
  •  TRE:Syk-15-tTEVp mini #1,#2
  •  TRE:Syk-3-tTEVp midi 
  •  A-15T9 midi
•  Sequencing 
  •  HyHEL10 H #2
  •  TRE:Syk-pTET:mRFP-TEVp (Syk3 read)
•  Pick CD79B colonies  
•  Inoculate midi Syk-6-tTEVp

 Wednesday's To Do's 

•  Golden gates, ligate, transform:
  •  CD79B-linker-G4
•  Midi
  •  A-12T12
•  Mini, digest, gel
  •  TRE:S-3-tT - don't digest this, we have a good one
  •  TRE:S-6-tT 
  •  TRE:S-9-tT 
  •  TRE:S-12-tT
•  Pick
  •  TRE:Syk-15-tTEVp
•  Innoculate midi:
  •  A15T9
  •  T:S3tT
•  Analyze sequencing
  •  CD79B-RFP good
  •  A-3T15 bad 
  •  A-9T15 bad
  •  A-15T9 good
  •  T:S-3-tTp (throw out new cultures + duplicate of mini)
•  Sequenicng
  •  A-3T15 #2 
  •  A-9T15 #2
  •  S-9-tT #1
  •  S-12-tT #1

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