Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Monday's to Dos

• Midiprep
  • something
• Golden gates
  • hEF1a:CD79B-pTet:RFP-Gal4VP16
  • pENTR Syk-15-tTEV
  • pENTR VRC01 light
  • pENTR VRC01 heavy
  • pENTR HyHEL-10 light
  • pENTR HyHEL-10 heavy
• Pick Colonies for mini
  • TRE:Syk-3-tTEV
  • TRE:Syk-6-tTEV
  • TRE:Syk-9-tTEV
  • TRE:Syk-12-tTEV
  • hEF1a:CD79A-9T15-G4
• Inoculate midis
  • From failed midiprep
    • hEF1a:Gal4
    • GGDonr
    • Linkers
  • Additional linkers
    • CD79A-6T9
    • CD79A-9T12

Tuesday's to Dos

• Transform golden gates
  • hEF1a:CD79B-pTet:RFP-Gal4VP16
  • pENTR Syk-15-tTEV
  • pENTR VRC01 light
  • pENTR VRC01 heavy
  • pENTR HyHEL-10 light
  • pENTR HyHEL-10 heavy
• Miniprep and digest
  • TRE:Syk-3-tTEV
  • TRE:Syk-6-tTEV
  • TRE:Syk-9-tTEV
  • TRE:Syk-12-tTEV
  • hEF1a:CD79A-9T15-G4
• Midiprep and digest
  • From yesterday's failed midipreps
    • hEF1a:Gal4
    • GGDonr
    • Linkers
  • Additional linkers
    • CD79A-6T9
    • CD79A-9T12
• Pick colonies for failed linkers
  • CD79A-3T15
  • CD79A-12T9
  • CD79A-15T9
• Send for sequencing
  • Resend 12T12 (1), also send 12T12 (2)
  • 9T15 (1) and (2)

Tuesday's to Dos

• Transform golden gates
  • hEF1a:CD79B-pTet:RFP-Gal4VP16
  • pENTR Syk-15-tTEV
  • pENTR VRC01 light
  • pENTR VRC01 heavy
  • pENTR HyHEL-10 light
  • pENTR HyHEL-10 heavy
• Miniprep and digest
  • TRE:Syk-3-tTEV
  • TRE:Syk-6-tTEV
  • TRE:Syk-9-tTEV
  • TRE:Syk-12-tTEV
  • hEF1a:CD79A-9T15-G4
• Midiprep and digest
  • From yesterday's failed midipreps
    • hEF1a:Gal4
    • GGDonr
    • Linkers
  • Additional linkers
    • CD79A-6T9
    • CD79A-9T12
• Inoculate midiprep cultures for successful CD79A linkers (based on sequencing data that we should get back today) and pick new colonies for not successful ones

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