User:Eric M. Walters/Notebook/Spring 2012/2012/03/27

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Continuing colony PCR of putative clones

Based on the strange results obtained/analyzed between Saturday/Monday, I'm going to do some more definitive tests on the streakouts.

  • 50-68°C gradient on gDNA and #31 (different patterns) with new upF/dnR primers (+:5.5 kb, -:nothing or nonspecific)
    • This should find me a good temperature for amplifying the whole fragment, and will let me know that the accBCDA insert is in the cell. I should be able to assert that growth means acsA has been knocked out as well, though there is potential for spontaneous loss of function.
  • accBCDA-F + old dnR primer (+:5 kb, -:nothing)
    • This will actually tell me that the accBCDA insert is where it should be. Hopefully has a better chance of working than the original upF/dnR primers, which are trouble.

Whole insert, new primers, temperature gradient

  • Just doing #31 and the negative control, they had significantly different bands before
  • 8 of each, between 50 and 68 degrees

10 μL reaction:

  • 5 μL GoTaq (85 in master mix)
  • 2 μL H2O (34)
  • 1 μL new acsA-up-F primer (17)
  • 1 μL new acsA-down-R primer (17)
  • 1 μL template (dilute 7002 gDNA or resuspended #31 in water)

Thermal cycler program:

  • 98°C 2:00
  • 98°C :30
  • 50-68°C :30
  • 72°C 5:00 (5 kb with GoTaq)
  • Goto 2 34x
  • 72°C 10:00


accBCDA to chromosome

  • 23 colonies, picked from what's available and struck out in patches on a 500 μM plate

10 μL reactions:

  • 5 μL GoTaq (125 in master mix)
  • 3 μL H2O (75)
  • 1 μL accBCDA-F primer (25)
  • 1 μL original acsA-down-R primer (25)

Thermal cycler program:

  • 98°C 2:00
  • 98°C :30
  • 54°C :30 (according to NEB calculator with Taq polymerase, 100 μM primers)
  • 72°C 5:45 (5.5 kb with GoTaq)
  • Goto 2 34x
  • 72°C 10:00