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From 2008.igem.org

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Revision as of 03:10, 3 June 2008

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Contents 1 Made Competent Cells 1.1 Seed Stock 1.2 Growth curve 1.3 Competent Cells 2 Discussion

Made Competent Cells

Grace, Margaret, Norman

Seed Stock

• Tested OD600 of 1 seed stock tube @ 16 hr after innoculation

• OD600 = 0.53244

• Separated each 1.8 mL seed stock tube to two 0.9 mL tubes

• Original cryotubes = 2 mL, which is not big enough for addition of glycerol later
• Next time, perhaps use other type of tube (cryotubes don't allow air for E. coli to grow)

• Incubated at 37C with shaking for additional 3 hrs

• Protocol suggests 23C but growth was too slow. Also, protocol is for Mach10 cells, which grows much faster.

• Added 158.8 ul glycerol to 6 seed stock tubes per compentency protocol
• Added 327.8 ul glycerol to 15 seed stock tubes (26.7% glycerol) per SC's recommendation

Growth curve

• Took one 0.9 mL seed stock tube prior to additional incubation and added to 90 mL SOB media
• Tested OD600 every hour for 90 mL SOB mixture and 250 mL SOB mixture made yesterday

(add table)

Competent Cells

• Competent cells prepared according to protocol based on Hanahan
• 100 ul competent cells were aliquoted into each tube and stored at -80C

(add table)

Discussion

Transform E.coli with PRL1383a tomorrow?

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

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