Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Monday's to Dos

•  inoculate cd79a 15t3, 15t15, 9t9 midis
•  analyze cd79a linker sequencing for previously failed linkers when it comes in
•  Golden Gate Syk-linker-tTEV linkers and transform gg
•  Re-PCR Q3-RFP-Q4
•  Transform:
  •  GG hEF1a:CD79B-pTet:RFP-Gal4VP16 with new RFP
  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-linker12-tTEVp golden gatestTEV
  •  
  Gel:
  •  digests
  •  aari diagnostic
•  Sequencing
  •  cd79a linkers
  •  pENTR RFP-IgM
•  Miniprep 3t15, 15t9, hef1a:gal4
•  midi linkers
•  Digest minis and midis
• • hEF1a:CD79A-9T15-G4
•  Inoculate midis
  •  From failed midiprep
    •  hEF1a:Gal4
    •  GGDonr
    •  Linkers
  •  Additional linkers
    •  CD79A-6T9
    •  CD79A-9T12

TuesdayMonday's to Dos

•  Midiprep
  •  something
   Golden 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
•  Pick Colonies for miniMiniprep and digest
  •  TRE:Syk-3-tTEV
  •  TRE:Syk-6-tTEV
  •  TRE:Syk-9-tTEV
  •  TRE:Syk-12-tTEV
  •  hEF1a:CD79A-9T15-G4
•  Inoculate midisMidiprep and digest
  •  From yesterday's failed midiprepmidipreps
    •  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