Haynes Lab:Notebook/CRISPR Editing/2014/07/08

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Retrying the ligation, won't leave overnight again.
Annealing gRNA oligos

3ul 100uM top oligo
3ul 100uM bottom oligo
2ul 10x annealing buffer
12ul H2O

20 ul reactions

boil and cool method
Aluminum foil lid on the beaker to prevent condensation
Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C).
Float the oligo mixtures in the boiling water for 10 min. Cover the beaker with aluminum foil to keep the air above the tube warm.
Turn off the heat source and allow the aluminum foil-covered water bath to slowly cool to room temperature (25°C) for several hours.


Label Backbone Insert
Bb 0 pX330 bb ctrl
1 pX330 g14
2 pX330 g5
3 pX330 g13
4 pX330 gn1T
5 pX330 gn1B
6 pX330 gn3T
7 pX330 gnBb B
Bb 5 pX335 bb ctrl
8 pX335 gn3T
9 pX335 gn1B
10 pX335 gn1T
11 pX335 gn3bB

Left for 1 hour, long transformation

Plated 100,000 cells into each well of two 6-well plates. Added G418 to final concentrations of 0, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, and 1500 μg/mL.

This is the second attempt because the first time G418 didn't kill the cells. We believe the drug was bad because it was expired by about 2 years.