BCR 2.0

Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

 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

 Tuesday's To Dos

• Re-innoculate midi
  • A-12T12
• Analyze sequencing data
  • 3T15 inconclusive 
  • VRC01L - probably right
  • resend seqeuncing
• Midiprep, digest, gel
  • A-12T12-bad yield
• Miniprep, digest, gel
  • TRE:S-6-tT
• Proteinase K kill, transform
  • Syk-15-tTEVp pENTR with TRE
• Pick colonies
  • TRE:S-3-tT
  • TRE:S-6-tT 
  • TRE:S-9-tT 
  • TRE:S-12-tT
• Sequence
  • VRC01L #2
  • TRE:Syk-6-tTEVp #1

Monday's To Dos

• 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

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