Cloning Strategy

CD79A Linker Optimization

Antibody Choices 

Tuesday's To Do's 

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

• 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
• Send for sequencing
  • Good minipreps
  • CD79B linkers from before (#1 from when we sent #2)
    • B9T12
    • B6T9

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