Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

STATUS TABLE

 To do's

•  Run gel pENTR HyHEL10 H
•  Mini
  
  •  A9T15 1,2
  •  B9T9 
•  Transform 
  •  B15T15
•  Mini, sequence
  •  A9T15 
  •  B9T9 
•  Midi:
  •  TRE:Syk-15-tTp
•  inoculate midi:
  •  B15T3
  •  T:S-9-TEVp
  •  T:S-12-TEVp
•  seq.
  •  B6T12 2
  •  pENTR HyHEL10 H 1
  •  B12T12 2
  •  A15T9 2
  •  B9T9 (8/12)
•  repeat seq.
  •  T:S-3-T 1
  •  T:S-6-T 1
  •  T:S-9-tT 2
  •  T:S-12-tT 2
  •  B3T15 1
  •  B9T15 1

Tuesday's To Do's 

•  Gel TRE:Syk-15-tTEVP -> send for sequencing 
•  digest, gel, pENTR HyHEL10 H -> send for sequencing 
•  sequencing:
  •  B6T9-1
  •  B9T12-1
•  innoculate midi:
  •  B15T3
  •  T:S-15-tT

•  inoculate mini:
  •  B9T9 - from plate
  •  A3T15 - from plate 
•  restreak/replate
  •  B15T9
  •  A9T15 - no white
  •  B15T15 
  •  B9T9 - only one white

:

•  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

Check on status:

• B9T12 - send for seq
• B6T9 - send for seq
• B12T9 - repeat seq
• B12T12 - repeat seq

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

Non-Lab To Do's 

•  Cytometry time 
•  Order Cumate 
•  Chicken lysozyme, gp120
•  Antibodies for antigen binding detection

Waiting on primers to do list:

• Bug Brian about ordering antibody testing supplies

Higher Level To Do's:

•  Decide on other antigen-variable regions we want to test  
•  Is gantenerumab proprietary and can we actually publish with it?

THINGS TO NOT FORGET TO DO:

• SYK-15tTEVP golden gate when we figure out what's wrong with the AarI
• 3T3 MIDI