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

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07/08/2014

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

Assemblies
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.

Ligate

Label Backbone Insert
Bb 0pX330bb ctrl
1pX330g14
2pX330g5
3pX330g13
4pX330gn1T
5pX330gn1B
6pX330gn3T
7pX330gnBb B
Bb 5pX335bb ctrl
8pX335gn3T
9pX335gn1B
10pX335gn1T
11pX335gn3bB



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.




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