Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Tuesday's To Do's

•  Gel for oligo annealing
•  Mix new annealing buffer
•  Anneal oligos
•  Design IgM Sequencing Primers
•  CD79B, gal4 PCR
•  Golden Gates:
  •  hEF1a:CD79B-pTET:mRFP-Gal4 golden gate
  •  TRE:Syk-15-tTEVp
•  Transform
  •  hEF1a:CD79B-pTET:mRFP-Gal4 GG
  •  TRE:Syk-15-tTEVp GG
•  TRE:Syk-pTET:mRFP-tTEVp sequencing 
•  Pick pENTR RFP-IgM Colony

Wednesday's to Dos

•  rePCR Gal4, CD79B
•  Gel PCRs
•  GG hEF1a:CD79B-pTet:RFP-Gal4VP16
•  Transform hEF1a:CD79B-pTet:RFP-Gal4VP16

Thursday's to Dos

•  Review Syk-RFP-tTEV Cloning (rePCR?)(Used wrong RFP for GG?) 
•  hEF1a:Gal4 pick new colony
•  pick hEF1a:CD79B-RFP-Gal4VP16 colonies (no red colonies present)
  
•  pick pENTR RFP-IgM Colonies
•  Innoculate midis of CDA linker 
•  Pick colonies of failed CDA linker constructs
•  Analyze TRE:Syk-pTet:RFP-tTEV Sequencing
•  Resend correct TRE:Syk-pTet:RFP-tTEV for sequencing

Friday'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
  •  TRE:Syk-linker-tTEVp golden gates
•  Gel:
  •  digests
  •  aari diagnostic
•  Sequencing
  •  cd79a linkers
  •  pENTR RFP-IgM
•  Miniprep 3t15, 15t9, hef1a:gal4
•  midi linkers
•  Digest minis and midis

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
•  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