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BCR 2.0

 

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Cloning Progress

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Timeline

Protocols

 

 

Cloning Strategy
CD79A Linker Optimization
Antibody Choices 

 

Expand
titleWednesday's To Do's
Wednesday's To Do's
  •  Flow cytometry with Brian (big grin)
  •  Analyze flow cytometry data
  •  Miniprep and verify, then sequence
    •  CD79A-RFP-G4 SDM
    •  CD79B-RFP-G4 SDM
  •  Midiprep (and verify?)
    •  pEXPR TRE:Syk
    •  hEF1a:CymR
    •  hEF1a-CuO:mKate
    •  CMV5-CuO:mKate
  •  LRs from Tuesday (that we probably won't have done because LR clonase shortage...)
  •  PCRs of mRFP and mIgM constant region for hEF1a:mRFP-CH (constant heavy chain)
  •  Order chicken lysozyme and gp120?
Tuesday's To Do's
  •  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?
    •  Review sequencing
      •  TRE:Syk-pTet:mRFP-TEV
      •  TRE:Syk-pTet:mRFP-tTEV
      •  pENTR CD79B-TCS-G4
  •  Add dox to cells
  •  Verify
    •  hEF1a:eYFP
    •  TRE:eYFP
    •  TRE:mKate
  •  Miniprep and verify
    •  CD79A-linker-G4 colonies (3T3, 3T9, 3T15)
    •  CD79A-RFP-G4 SDM colonies
    •  hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate
  •  Send for sequencing
    •  CD79A-linker-G4 colonies
    •  CD79A-RFP-G4 SDM colonies
  •  Re-transform SDM reactions to hopefully get more colonies to choose from?
  •  Start midiprep cultures?
    •  hEF1a:CymR
    •  hEF1a-CuO:mKate
    •  CMV5-CuO:mKate
    •  pEXPR TRE:Syk?
  •  Pick colonies for miniprep cultures
    •  pEXPR TRE:Syk?
    •  CD79A-RFP-G4 SDM
    •  CD79B-RFP-G4 SDM
  •  LR - probably can't do today because no LR clonase
    •  EGSH:mKate
    •  hEf1a:VgEcR-2A-RXR
    •  hEF1a:mKate
    •  hEF1a:Gal4VP16 (remember which pDEST)
    •  hEF1a:CD79B-TCS-Gal4VP16 (ONLY after sequencing pENTR CD79B-TCS-Gal4VP16)
Monday's To Do's 
  •  make BCR enzyme stock
  •  digest, gel:
    •  pENTR mKate
    •  hEF1a:CD79B-TCS-G4
    •  hEF1a:Gal4VP16
  •  sequence:
  •  transform:
    •  TRE:syk because we don't have a midi
    •  Re-transform CD79A-TCS-G4 and CD79B-TCS-G4 SDM Golden Gates 
  •  pick more CD79A linker colonies (all old DNA is not good)
  •  pick CD79A SDM colony 
  •  pick hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate

 

CMV5-CuO 715:

hEF1a-CuO 715:09

CymR-CuO 710:09


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

 

BCR 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?