Team:Hawaii/Notebook/2008-06- 2

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* Competent cells prepared according to [[Team:Hawaii/Make_Competent_E._Coli | protocol]] based on Hanahan  
* Competent cells prepared according to [[Team:Hawaii/Make_Competent_E._Coli | protocol]] based on Hanahan  
* 100 ul competent cells were aliquoted into each tube and stored at -80C
* 100 ul competent cells were aliquoted into each tube and stored at -80C
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(add table)
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{| border="1"
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|+ '''OD readings for competent cells, with CCMB80 added'''
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! Tube !! OD600 !! OD550
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|-
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! 1
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| 1.1936 || 1.2488
 +
|-
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! 2
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|1.1182
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|1.1718
 +
|-
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! 3
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|1.1289
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|1.1849
 +
|-
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! 4
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|1.0059
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|1.0679
 +
|}
 +
'' Note: Desired OD600 = 1.0-1.5''
== Discussion ==
== Discussion ==

Revision as of 03:22, 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
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?


[http://manoa.hawaii.edu/ Sponsor_UHM.gif][http://manoa.hawaii.edu/ovcrge/ Sponsor_OVCRGE.gif][http://www.ctahr.hawaii.edu Sponsor_CTAHR.gif]

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