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Team:Georgia State/Notebook
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Revision as of 12:19, 24 August 2010 by Zacstewart (Talk | contribs)
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.
