Team:Hawaii/Notebook/2008-06- 2

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==== Growth of DH5-&alpha from seed stock====
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==== 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
Added one 0.9 mL seed stock to 90 mL SOB and checked growth (OD550 & OD600) every hour
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!width="100"|Time
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!width="100"|OD 600
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! 1 hr
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|reading 600
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|reading 550
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Revision as of 04:38, 3 June 2008

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Contents

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-α (citation?)


Growth Curve of DH5-α in SOB
Time OD for 90 mL solution OD for 250 mL solution
11AM OD600 = 0.007 OD600 = 0.12
12PM OD600 = 0.01089 OD600 = 0.22
1PM OD600 = 0.05371 OD600 = 0.48099, OD550 = 0.5303
2PM OD600 = 0.15752, OD550 = 0.17463 OD600 = 0.57673, OD550 = 0.63531
3PM OD600 = 0.33753, OD550 = 0.37611 n/a



Growth of DH5-α from seed stock

Added one 0.9 mL seed stock to 90 mL SOB and checked growth (OD550 & OD600) every hour

Growth of DH5-α from seed stock
Time OD 600 OD 550
1 hr 0.0272 0.03005
2 hr reading 600 reading 550
3 hr reading 600 reading 550
4 hr reading 600 reading 550



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
  • 100 ul competent cells were aliquoted into each tube and stored at -80C


OD readings for competent cells, with CCMB80 added
Tube OD600 OD550
1 1.1936 1.2488
2 1.1182 1.1718
3 1.1289 1.1849
4 1.0059 1.0679

Note: Desired OD600 = 1.0-1.5

Discussion

Transform E.coli with PRL1383a tomorrow?


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