Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

:

•  mini pENTR hEF1a-LacO, pENTR LacI

•  mini culture pENTR mKate

•  mini pENTR mKate

•  verification digests, gels

•  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)

Sequencing account information:

LR EGSH:mKate, hEf1a:VgEcR-2A-RXR

restreak TRE:Syk-pTET:mRFP-(t)TEVp

sequence TRE:syk-ptet:mrfp-(t)TEVP

TRE:Syk mini transformation

MAV1212 transformation 

Thursday's To Do's

•  Kill LRs
•  DpnI digest SDM
•  SDM CD79B-RFP-G4 PCR purification
•  Anneal Syk Linker oligos 
•  Transform:
  •  hEF1a:CymR, hEF1a-CuO:mKate, CMV5-CuO:mKate LRs
  •  SDM CD79B-RFP-G4
  •  pENTR mKate
•  pick pENTR LacI, pENTR hEF1a-LacO colonies
•  midi TRE:Gal4VP16, TRE:mKate, hEF1a:LacI
•  digest, gel pLac:mKate LR #2
•  grow up pLac:mKate midi culture

Wednesday's To Do's

•  SDM cd79b-ptet:mrfp-gal4vp16 - Christian
•  Innoculate Midi TRE:gal4VP16 (2nd transformation #1) -  Lyla 
•  Transform: 
  •  pENTR LacI and pENTR pLac - Lyla 
•  Miniprep, digest, gel 
  •  TRE:Syk-pTet:mRFP-TEVp
  •  pLac:mKate
  •  TRE:gal4vp16 #2 (should be in unverified mini/midi in 4C) - see cloning progress 06/26
  •  rerun digest/gel from yesterday (TRE:mKate and hEF1a:LacI LR1 #2,3 with SalI and ApaI resp.)
•  LR hef1a:cymR and hEF1aCuO:mkate (look to see if CMV5CuO is in lab promoter box - report number to Brian) - Lyla 

CMV5-CuO 715:

hEF1a-CuO 715:09

CymR-CuO 710:09

Geneious To Do List:

•  

  SDM primers: CD79B, Gal4VP16, TEVp

•  TRE:gal4vp16 sequencing primers
•  TRE:Syk-mRFP-(t)TEVp sequening primers - Kathryn
•  Antibody cloning for chicken lysozyme - Kathryn 
•  hef1a sequencing primers

BCR 2.0 To Do
^^^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?