Team:Hawaii/Notebook/2008-06- 2

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
 * Growth until OD = 0.3 - 0.6, cells then made competant
 * OD600 is ideal for measuring Mach10 growth; OD550 should be used for DH5-&alpha; (citation?)

Growth of DH5-&alpha; from seed stock
Added one 0.9 mL seed stock to 90 mL SOB and checked growth (OD550 & OD600) every hour

Competent Cells

 * Competent cells prepared according to protocol based on Hanahan
 * Competent cells were made from stocks w/ OD600 = 0.33753 and 0.57673
 * 250 ul chilled CCMB80 was added to each 50 ul aliquot of OD600=~0.6 to get final OD600 = 1.0-1.5

 Note: Desired OD600 = 1.0-1.5
 * 100 ul competent cells were aliquoted into each tube and stored at -80C

Discussion

 * Competency test tomorrow morning
 * Test transformation of E. coli w/ PRL1383a Thursday if cells made are competent
 * Okay to grow up E. coli cells (for inducing competency) from seed stock?

Quote of the Day
"You know what's ironic? If I take my shirt off, it'll just get hotter." -An anonymous friend