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BCR 2.0

 

Cloning Progress

Experiments

Parts Tree

Parts Library

Timeline

Cloning Workflow

Protocols

 

 

Cloning Strategy
CD79A Linker Optimization
Antibody Choices 

 

2015-07-10 To Do's 
  • Sodium Azide Solution
  • Make Kan X-gal plates
  • (restart) Midi:
  • digest, gel:
  • transform:
    • TRE:Syk-15-tTEVp golden gate
    • Golden gate donor mini product
    • LRs:
      • EGSH:mKate
      • hEf1a:VgEcR-2A-RXR
      • TRE:mKate
      • hEF1a:Gal4VP16 (remember which pDEST)
      • hEF1a:CD79B-TCS-Gal4VP16 
      • hEF1a:tagBFP
Datestuff
2015-07-09
  • gel verify:
  • Pick colonies for midi
  • PCR purify TRE:Syk-15-tTEVp
  • Golden gate TRE:Syk-15-tTEVp
  • LRs:
    • EGSH:mKate
    • hEf1a:VgEcR-2A-RXR
    • TRE:mKate
    • hEF1a:Gal4VP16 (remember which pDEST)
    • hEF1a:CD79B-TCS-Gal4VP16 
    • hEF1a:tagBFP
2015-07-08
  • Flow cytometry with Brian (big grin)
  • Order chicken lysozyme and gp120?
  • Non-lab to do's

      • Design (or find?) sequencing primers for mIgM CH (constant region)
      • Plan experiment to test gantenerumab binding to beta-amyloid
      • Leader sequences for ordering gBlocks of variable regions?

CMV5-CuO 715:

hEF1a-CuO 715:09

CymR-CuO 710:09

 


Geneious To Do List:
  • Antibody cloning for chicken lysozyme - Kathryn 
  • hef1a sequencing primersBCR 2.0 To Do

^^^Email: bcrpeeps@mit.edu

^^^Pass: grapefruit

 

 

Higher Level To Do's:
  • Decide on other antigen-variable regions we want to test  
  • Other experimental planning (on experimental page)
  • Parts list (excel sheet detailing specs/locations of parts)
  • How do we do site directed mutagenesis?
  • Literature research: 
    • kozak sequences for heavy and light chains and CD79s
    • truncated TEVp
    • other antigens to detect using BCR (we were thinking of small molecule detection)
    • inducible promoters (we were considering LacI - an inducible repressor - but we want to look into other options first) 
  • Cloning planning 
    • hEF1a:Syk-pTet:mRFP-TEVp
    • variable region 
  • Is gantenerumab proprietary and can we actually publish with it?

 

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