Notebook

Sunday, July 31^st

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