Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Saturday's To Do's

• Kill LR (TRE:Syk-15-tTEVp)
• Take out plates + colonies 

Friday's To Do's

• Midi, digest, gel
  • TRE:Syk-6-tTEVp
• 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

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

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