Team:Northwestern/07 31

Notebook

Sunday, July 31st

Tasks:

Sara & Sam

  • Reran (7/29) PCR Linearization of the Tet Backbone for Cas9 Gibson +DMSO
    • 4 tubes of the following recipe in order to increase concentration resulting from gel extract:
      • 10 uL water
      • 0.5 uL DMSO (used to increase purity of results)
      • 1.0 uL Tet template (from miniprep)
      • 0.5 uL 10uM fwd primer
      • 0.5 uL 10uM rev primer
      • 12.5 uL OneTaq Master Mix
      • Total: 25 uL
      • 95°C (2:00) | 95°C (0:07),  60°C (0:10),  72°C (1:06) [10 cycles] | 95°C (0:07), 50°C (0:10), 72°C (1:06) [18 cycles] | 72°C (5:00), 4°C (20:00)
    • DpnI digest: 1 uL of DpnI added to each of the 4 tubes. Incubated in the 37 for one hour
    • Ran gel
      • Loaded 20 uL of each of the 4 tubes of product into each well (we didn’t want to overload), and the other 5 uL into the next 4
      • All 4 are the same product and look to be about the right size, accounting for the wonkiness of the gel
    • Gel extracted of the Linearized Tet Backbone- Gibson
      • Concentration: 41.6 ng/uL
  • Reran PCR of GFP and mCherry
    • 25 uL OneTaq
    • 1 uL diluted 10 mM f primer
    • 1 uL diluted 10 mM r primer
    • 1 uL GFP/mCherry
    • 21 uL dH20
    • 1 uL DMSO
    • Ran 2 tubes for each GFP and mCherry at 50 uL volume, per Patrick’s advice
    • 95°C (2:00) | 95°C (0:07), 51°C (0:10), 72°C (0:43) | 72°C (5:00)
  • DpnI digest
    • Incubated at 37°C for 2 hours, then overnight
    • 1 uL per 50 uL PCR reaction

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