Page History

To Do

•  Tuesday----------------------------------------------------------------------------------------------------------
•  Immunostaining Microscopy protocol
•  Immunostaining Flow Cytometry protocol
•  Western Blot protocol
•  Analyze PCR sequencing
•  Gel extract new Syk-eYFP PCRs
•  Midiprep CD79Bs
•  Transform TRE Gal4VP16
•  Trasfect for membrane localization test
•  Monday-----------------------------------------------------------------------------------------------------------
•  Double check basic plasmids
•  Flesh out initial experiments (CR, KRB)
•  PCR Syk & eYFP
•  Sequence Syk and eYFP PCR Products (AS)
•  PCR Screen Syk eYFP (KRB, CR)
•  Analyze Syk eYFP gel (CR, KRB)
•  Digest and gel Lyn pENTRs and CD79B pEXPRs (EE)
•  Friday--------------------------------------------------------------------------------------------------------------
•  Verify CD79B-stop LR components, add to wiki
•  Grow up liquid cultures for miniprep
  •  GG: Lyn-stop
  •  GG: Lyn-mKate
  •  GG: Lyn-TEVp
  •  LR: CD79B+stop
  •  pDEST 1-2
  •  pDEST 2-3 -> from amp only plate, then sequence
•  Digest and gel
  •  pEXPR hEF1a: CD79B-TCS-G4
  •  pENTR Syk-eYFP
•  Submit for sequencing Syk-eYFP samples (based on gel)
•  Midiprep
  •  pEXPR hEF1a: Gmab H
  •  pEXPR hEF1a: Gmab L
•  Streak Lyn plates?
•  Miniprep pEXPR hEF1a: CD79B-TCS-G4
•  Additional parts? -> primers, etc.
•  Thursday---------------------------------------------------------------------------------------------------------
•  Transform and plate
  •  LR: CD79B+STOP (amp)
  •  pDEST 1-2 (amp/chlor)
  •  pDEST 2-3 (amp/chlor)
  •  GG: Lyn+stop (ggdonr)
  •  GG: Lyn-mKate (ggdonr)
  •  GG: Lyn-TEVp (ggdonr)
•  Make amp/chlor plates (AS)
•  Miniprep (EE)
  •  pEXPR CD79B-TCS-Gal4VP16
  •  pENTR Syk-eYFP
  •  whatever's in the incubator with these
•  Sequence pENTR Syk-eYFP
•  Digest and Gel
  •  pEXPR CD79B-TCS-Gal4VP16
  •  pENTR Syk-eYFP?
•  Add Gmab gel to wiki (KRB)
•  Review 3D structure linker information (KRB, CR)
•  Lyn plates out of incubator
•  GmabH&L=>Verified Plasmids (CR)
•  Plasmid hunting: (CR)
  •  hEF1a: Gal4VP16
  •  TRE: Gal4VP16
•  Grow up for midipreps:
  •  GmabH
  •  GmabL
•  Wednesday------------------------------------------------------------------------------------------------------
•  Labwork
  •  Midiprep four plasmids
  •  Miniprep Gmab H and Gmab L
  •  Restriction Map H&L
  •  Transform and plate Lyn Golden Gates, Donr 2-3?
  •  Pick colonies (CD79B-TCS-Gal4VP16, Syk-eYFP GG) (also others
  •  LR CD79B-STOP
•  Wiki/Design
  •  Update and expand on all experiments
  •  add gel image to wiki
  •  how we chose linker links on wii