Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Tuesday's To Do's 

•  Gel pENTR HyHEL10H TRE:Syk-15-tTEVP -> send for sequencing 
•  digest, gel, pENTR HyHEL10 H -> send for sequencing 
•  Midiinnoculate midi:
  •  B15T3

•  B3T3
  •  B3T9

• •  B6T6
  •  B9T3
  •  B9T6
•  Mini:
  •  iowrf

• inoculate mini:
  •  B15T9 - from mini culture
  •  B9T9 - from plate
  •  B15T15 - from plate
  •  A3T15 - from plate 
  •  A9T15 - from plate 

Monday's To Do's

READ THIS:  I didn't get around to going into lab until late so I put the midipreps in around 1am.  Don't midiprep them until the afternoon, otherwise we might get low concentrations.  Minipreps and plates were also put in around 1am-2am.  Also the parts pages for h:CD79A-...-G4 and h:CD79B-...-G4 have been updated with the actual status of each construct.

•  Take plates out of the incubator
•  Miniprep
  •  From before (in the fridge)
    •  pENTR HyHEL10 Heavy
    •  B-9T15 
  •  New cultures
    •  A-3T15
    •  A-9T15
    •  A-15T9 (only one tube)
    •  TRE:Syk-3-TEVp
    •  TRE:Syk-6-TEVp
    •  TRE:Syk-9-TEVp
    •  TRE:Syk-12-TEVp
    •  B-3T15
    •  B-6T12
    •  B-15T9
    •  TRE:Syk-15-tTEVp
•  Pick colonies (plates currently in the incubator - were restreaked or replated)
  •  B-9T9 - replated
  •  B-15T15 - restreaked
•  Set up repeat sequencing order (waiting on QC info to come in)
  •  B-12T9
  •  B-12T12
•  TRE:Syk-9-tTEVp (or just send in sample #2 - this has been sent for sequencing twice now with bad reads)
  •  TRE:Syk-12-tTEVp (or just send in sample #2 - this has been sent for sequencing twice now with bad reads)
   Send for sequencing
  •  Good minipreps
  •  CD79B linkers from before (#1 from when we sent #2)
    •  B9T12
    •  B6T9
  •  Maybe TRE:Syk-9-tTEVp #2 and TRE:Syk-12-tTEVp #2
•  Seed for transfection and split
  
•  Midiprep
  •  B3T3
  •  B3T9
  •  B6T6
  •  B9T3
  •  B9T6
  •  B12T6
•  Start midiprep culture (wasn't midiprepped before because there were 7 midipreps to do total and the centrifuge does 6)
  •  B15T3

Sunday's To Do's

•  Take out plates
•  Pick colonies
  •  Plated 8/8
    •  A-15T9
    •  TRE:Syk-3-TEVp
    •  TRE:Syk-6-TEVp
    •  TRE:Syk-9-TEVp
    •  TRE:Syk-12-TEVp
    •  B-3T15
    •  B-9T9
    •  TRE:Syk-15-tTEVp
  •  Streaked or restreaked 8/8
    •  A-3T15
    •  B-15T15
    •  B-15T9
    •  B-6T12
  •  Plated previously
    •  A-9T15
•  Start midiprep cultures
  •  
    B3T3
  •  
    B3T9
  •  B6T6
  •  B9T3
  •  B9T6
  •  B12T6
  •  B15T3

Saturday's To Do's

•  Kill LR (TRE:Syk-15-tTEVp)
•  Take out plates + colonies 
•  Transform
  •  hEF1a:CD79A-15T9-G4 (because even though we midi'ed it, sequencing was bad)
  •  TRE:Syk-linker-TEVp (3, 6, 9, 12)
  •  hEF1a:CD79B-3T15-G4
  •  hEF1a:CD79B-9T9-G4
  •  TRE:Syk-15-tTEVp
•  Streaked out
  •  A-3T15
  •  B-15T15
  •  B-15T9
  •  B-6T12

Sequencing account information:
^^^Email: bcrpeeps@mit.edu

^^^Pass: grapefruit

Non-Lab To Do's 

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

Waiting on primers to do list:

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