Team:Hawaii/Notebook/2008-06- 2

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(Difference between revisions)
(New page: {{Team:Hawaii/Header}} == Made Competent Cells == '''Grace, Margaret, Norman''' ====Seed Stock==== * Tested OD600 of 1 seed stock tube @ 16 hr after innoculation :* OD600 = 0.53244 * Sep...)
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* Took one 0.9 mL seed stock tube prior to additional incubation and added to 90 mL SOB media
* 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
* 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?)
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(add table)
+
{| border="1"
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|+ '''Growth Curve of DH5-alpha in SOB'''
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! Time !! OD for 90 mL solution !! OD for 250 mL solution
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|-
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! 11AM
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| OD600 = 0.007 || OD600 = 0.12
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|-
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! 12PM
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|OD600 = 0.01089
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|OD600 = 0.22
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|-
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! 1PM
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| OD600 = 0.05371 || OD600 = 0.48099, OD550 = 0.5303
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|-
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! 2PM
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| OD600 = 0.15752, OD550 = 0.17463 || OD600 = 0.57673, OD550 = 0.63531
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|-
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! 3PM
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| OD600 = 0.33753, OD550 = 0.37611 || n/a
 +
|}
====Competent Cells====
====Competent Cells====

Revision as of 03:18, 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-alpha (citation?)
Growth Curve of DH5-alpha 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

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?


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