Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

Wednesday's To Do's

•  Flow cytometry with Brian
•  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:
  •  TRE:syk-ptet:mrfp-(t)TEVP
  •  hEF1a:CD79B-TCS-G4

•  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 

:

•  Midiprep (from freezer)
  •  S9TEV
  •  B15T15
•  Golden Gate
  •  B9T9
  •  B15T9
•  LR
  •  hEF1a-CuO:Gmab H
  •  hEF1a-CuO:Gmab L
  •  hEF1a-CuO:CD79A
  •  hEF1a-CuO:CD79B
  •  hEF1a-CuO:Lyn
•  Digest all of the Syk-TEVp and Syk-tTEVp midis just to be sure (since there's an internal cut site that didn't get read in sequencing)

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

Sequencing account information:

^^^Email: bcrpeeps@mit.edu

^^^Pass: grapefruit

## Digest tTEVp and TEVp midis just to be sure (since there's an internal cut site)

Non-Lab To Do's 

•  Bug Brian about ordering antibody testing supplies

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?