Haynes Lab:Notebook/CRISPR Editing/2014/07/04

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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.

PCR of gBlock of mCh donor sequence with stop codons at three spots. After PCR with three sets of primers, will have three donor sequences each with two stop codons in different locations.
Ran 2-100ul PCR reactions for each primer set, 51°C annealing temp, 30s extension time.

Digest of pX330 and pX335. 4ug of each in 80ul reaction, 4ul BbsI. Left at 37°C for about 25 minutes. gel extracted

Sample Read# 260 280 260/280 ng/µL total ug %recovery
pX330 3.70E-02 1.80E-02 2.052 36.788 1.8394 0.45985
pX335 4.70E-02 2.40E-02 1.918 46.529 2.32645 0.5816125

also ran mCh donor sequence on the gel, didn't see any bands but it's possible I didn't have enough EtBr because all my bands were faint. The bands should be pretty small so even though they're PCR products, they still might be faint. Didn't think to soak the gel. will rerun

Calculated volume for 50ng of each backbone. Added the volume of annealed inserts that karmella added in the protocol I followed. I'm not sure if I should try to vary the volume but that would give me tons of reactions. This can be an option in the future if I have trouble ligating these.

' pX330 pX335
insert 0.5 0.5
backbone 1.33 1.06
2x ligase buffer 5 5
ligase 1 1
water 2.15 2.5
Total 10 10

Backbone Insert
pX330 bb ctrl
pX330 g14
pX330 g5
pX330 g13
pX330 gn1T
pX330 gn1B
pX330 gn3T
pX330 gnBb B
pX335 bb ctrl
pX335 gn3T
pX335 gn1B
pX335 gn1T
pX335 gn3bB