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BCR 2.0 0

Switchable Antigen-Sensing System (S.A.S.S.)

 

Center

Cloning Progress

Experiments

Parts Tree

Parts Library

Timeline

Cloning Workflow

Protocols

Papers & References

 

Cloning Strategy
CD79A Linker Optimization
Antibody Choices 

STATUS TABLE

 

 To do's:
  •  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:
^^^Email: bcrpeeps@mit.edu

^^^Pass: grapefruit

 

 

Experimental Progress:

Cloning Progress
Parts Tree
Cloning Strategy
Experiments
Linker Optimization
Antibody Choices 

 

Useful Links: 

Protocols
Immediate To Do's:
Expand
titleLinker optimization
TaskClaimed byDate CompletedOther Notes
CD79A/B, Gal4VP16, RFP PCR try3CRApr 10

 

Gel verification of try3 PCR productLAApr 10RFP and Gal4 worked, CD79s didn't - we probably need to redesign primers
Redesign primersLA + CRApr 11CD79A F and R and CD79B F primers were redesigned and ordered
AarI golden gate of CD79A constructLAApr 30 
Take out CD79A golden gate platesLAMay 2 

Make LB

CRMay 4 

CD79A golden gate liquid cultures

LAMay 4

Cultures in at 6pm - need to be taken out by 10/11 am May 5

in 215 shaking incubator

Take out CD79A golden gate liquid culturesLAMay 5In 4C

CD79A golden gate minipreps

LAMay 6In yellow rack in 4C - need to be nanodropped

CD79A golden gate restriction verification

CRMay 6Some promising constructs
Send promising CD79A golden gates for sequencing   
  • CD79A linker golden gates
   
Redesign CD79B reverse PCR primerCRMay 24 
Expand
titleVariable region construction
TaskClaimed byDate CompletedOther Notes
Order primers for PCRing constant region  primers on geneious
    

Christian is Confused so he is using this to do list

Day

List

Monday

  •  
    Establish HEK293 Cell Line from frozen stock

Tuesday

  •  
    None

Wednesday

  •  
    None

Thursday

  •  
    None

Friday

  •  
    Inoculate 5mL Cultures
  •  
    Re-Transform H:Lyn, T:Lyn-TEV, H:CDB-TCS-G4
  •  
    PCR CD79B
  •  
    Gel CD79B
  •  
    Sequence H:CDA-RFP-G4
Saturday
  •  
    Inoculate Midipreps
  •  
    Prepare Cryostocks
  •  
    Pick Colonies => Midipreps & 5mL for Glycerol Stocks
  •  
    Pour New Gels
  •  
    Gel Extract CD79B
  •  
    GG H:CDB-RFP-G4
  •  
    Check for Sequencing for H:CDA-RFP-G4
  •  
    Fix Colonies picked 05/29
  •  
    Check HEK Cells

Sunday

  •  
    Split HEK293FT Cells

## 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:
  •  Finish Discussing Figures for Experiment list Decide on other antigen-variable regions we want to test 
  •  Syk-Lyn experiment design 
  •  Receptor dilution experiment design
  •  Other experimental planning
  •  Is gantenerumab proprietary and can we actually publish with it?