Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Today's To Do's

•  restreak TRE:Syk-mRFP-(t)TEVp - eric 
•  miniprep digest verify TRE:Syk-mRFP-(t)TEVp
•  transform Transform: 
  •  pENTR LacI and pENTR pLac
•  pick hEF1a:LacI, TRE:mKate, pLac:mKate Miniprep, digest, gel hEF1a:LacI, TRE:mKate,
  •  TRE:Syk-pTet:mRFP-TEVp
  •  pLac:mKate
  LR2
  •  
  miniprep TRE:gal4 transformation 2 
•  digest, gel verify TRE gal4 transformation 2
•  split cells - Lyla 
•  pLac:mKate pick LR colonies - Lyla 
•  LR hef1a:cymR and cmv5CuO:mkate
•  ScaI to lab stock

Wednesday's To Do's

•  Transform: 
  •  LR hef1a:cymR and cmv5CuO:mkate
  •  pENTR LacI and pENTR pLac
•  Miniprep, digest, gel 
  •  TRE:Syk-pTet:mRFP-TEVp
  •  pLac:mKate

• • TRE:gal4vp16 #2 (should be in unverfied mini/midi in 4C) - see cloning progress 06/26
  •  rerun digest/gel from yesterday (TRE:gal4vp16 with XhoI, 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)
  
•  Split HEK293

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
•  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