Team:Georgia State/Notebook

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Contents

Notebook

August 12, 2010

  • Reading about P. pastoris as vaccine vector
  • Started engineering of plasmid

Joe

  • Inoculated LB+Ampicillin broth with glycerol stock of E. coli containing GFP
  • Located in 37°C incubator in Crow lab.

August 8, 2010

  • Centrifuge P. pastoris to prep for electroporation
  • Competent cells prepared, ready to pulse
  • Transformed/electroporated P. pastoris with 9A and GFP

August 9, 2010

Joe

  • Prepared YPD/Hepes
  • Prepared 10mL of DTT
  • Inoculated 2 1L flasks containing 250mL YPD with 500mL of P. pastoris from overnight culture. Stored in -80°C room at 30°C in shaker.
  • Protocol modifications for P. pastoris transformation are in notebook

August 8, 2010

Joe

  • Prepared YPD+sorbitol
    • 10g yeast extract
    • 20g peptone
    • 182.2g sorbitol
    • 900ml H20
  • Inoculated 250ml flash of YPD with P. pastoris from 6-29-10 culture in SAB
    • added 500ml of SAB culture into ≈100-125ml YPD, stored in shaker in 30°C incubator

August 5, 2010

  • 12E SpeI protocol, PstI protocol
  • 9A PstI proocol
  • 9A was purified as the Xba I previously used on it was not fast digest
  • Gel extraction of 9A and 12E

August 4, 2010

Virginia 12E plasmid extraction from 5-min kit. Lysis solution and wash buffer taken from eppendorf kit needs to be confirmed on Nano Drop.

August 3, 2010

Joe, Virginia

  • Tried to extract 12E plasmid with Jetgene kit starting with cells stored in resuspension buffer. (Started with step 2). Cell solution was very viscous. Difficult to pipette. Did not form a pellet in step 4.
  • Inoculated LB+Ampicillin with 12E and stored at 37°C from glycerol stock in -80°C.
    • Will try again with fresh cells
    • Ran out of buffer in Jetgene kit.

July 22, 2010

Virginia

  • Prep LB, YPD plate solution
  • Prep NEB 2+3 buffer solutions

July 20, 2010

Melissa, Alykhan

  • Prepared tris HCl for buffer solutions

July 15, 2010

Virginia, Angie, Alykhan, Joe

  • Cut plasmid and ligated
  • P. pastoris cells ready in -80°C

July 14, 2010

Virginia, Joe, Angie, Alykhan

  • 10 glycerol stocks of 12E
  • P. pasoris competent cells
  • Started, ready at 2pm
  • Plamid resuspension buffer made
  • LB agar aliquots
    • warm in water bath
    • one 10mL tube for 1L agar
  • extraction of 12E+9A plasmid parts: white tubes in freezer

July 13, 2010

Virginia

  • 10 glycerol stocks of each
    • P. pastoris
    • 9A E. coli
  • Inoculated 12E broth for plasmid extract and glycerol stocks
  • Plate of P. pastoris for quality control

July 8, 2010

Angie, Kendra, Melissa, Nishedh, Alykhan

  • Diluted pichia cells in a volume of 50mL to a OD600 = .186+.201
  • Transform pars 12E and 12M into E. coli

July 7, 2010

Joe, Kendra, Angie

  • Replated colonies from 9A RFP plate (3 plates).
  • Colonies on broth 100 µL for 9A but no fluorescence observed
  • 9A survivor colonies replated onto 10 µg/mL ampicillin plates and incubated at 30°C
  • Mother plate in fridge
  • Made 1000mL YPD
  • Growing P. anomala in YPD broth for competent cells protocol

July 6, 2010

Dan

  • Replated E. coli culture

Alykhan

  • Transformation 9A, 8I (digested) & GFP.

July 2, 2010

Alykhan and Virginia

  • DNA purification and ligation
  • Replated E. coli culture

July 1, 2010

Joe

  • Plated transformed cells containing either GFP (control) or iGEM part onto LB plates with 10µg/mL ampicillin (2 plates each, 10mL or 100mL)
  • Also plated ≈100mL (remaining amount) of cells onto LB plates not containing ampicillin.
  • Spread plates with hockey stick and placed in 37°C at 7:35.