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

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