Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Sunday'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)
•  Miniprep (if you can find 2mL tubes...)
  •  pENTR HyHEL10 Heavy
  •  hEF1a:CD79B-3T15-G4
  •  hEF1a:CD79B-9T9-G4
  • 9T15-G4 
•  Pick colonies Midi, digest, gel
  •  Plated 8/8
    •  A-15T9
    •  TRE:Syk-
    15-tTEVp

Friday's To Do's

• • • 3-TEVp
    •  TRE:Syk-6-
    tTEVp
   Mini, digest
  
  •  B-3T3-G4
  •  
    • TEVp
    •  TRE:Syk-
    15
    • 9-
    tTEVp
   Mini, sequence
   Golden Gate, transform
  
  • • TEVp
    •  TRE:Syk-12-TEVp
    •  B-
    3T9-G4
    • 3T15
    •  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
• • • 9T9
    •  TRE:Syk-15-tTEVp
  •  Plated previously
    •  A-3T15
    •  A-9T15
    •  B-15T15
•  Start midiprep cultures
  •  
    B3T3
  •  
    B3T9
  •  B6T6
  •  B9T3
  •  B9T6
  •  B12T6
  •  B15T9

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
•  Streak onto new plate (some white colonies hidden with red ones)
  
  •  hEF1a:CD79B-6T12-G4
  •  hEF1a:CD79B-15T9-G4 (restreak and replate)
•  Pick colonies Mini, Midi, digest, gel
  •  pENTR HyHEL10 Heavy
  •  hEF1a:CD79B-9T15-G4

Thursday's To Do's 

•  Inoculate midi Syk-6
  •  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  
• • -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

Non-Lab To Do's 

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

Waiting on primers to do list:

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