Haynes Lab:Notebook/CRISPR Editing/2014/09/24

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09/24/2014

Ran 4x50ul PCR reactions of gRNA 20 and gRNA 29 +dox samples 1 and 2 on a gel. All had one band, mostly high yields, one of gRNA29 sample 1 had a lower yield.

Set up four sets of annealing reactions for Luc14 samples, only annealed two of them, then cut all of them. One set was 0.5ul of Nuclease for 1hour, other set was 1ul for 30 minutes (paused the other one at about 28 minutes left in the incubation. This will tell me if nuclease is randomly cutting (if it cuts PCR product that wasn't annealed) and if reducing the time or the amount of nuclease will get rid of the smear I saw yesterday.
Purify the gRNA 20 and 29 +dox samples using genelute PCR kit. Elute in 50ul elution solution.

Sample Read# 260 280 260/280 ng/µL
gRNA 20 +dox 1 0.204 0.112 1.827 203.785
gRNA 20 +dox 2 0.179 0.096 1.863 178.784
gRNA 29 +dox 1 0.153 0.088 1.736 152.76
gRNA 29 +dox 2 0.196 0.106 1.848 195.683


Gel matches the PCR yields.

Gel of test shows that skipping the annealing step does not really modify the results, which makes sense because they're PCR reactions so they've already been denatured and annealed.
It also doesn't seem like reducing the amount of nuclease or digestion time gets rid of the smear but I think I can see enough cutting that it's worth doing the samples.
Annealed the samples for gRNA20 +dox 1 and 2, gRNA 20 Luc14, gRNA 29 +dox 1 and 2, gRNA 29 Luc14, gRNA 27 Luc 14 (this was supposed to be a no-cutting control but I think it's actually cutting)
Put at -20 for about an hour
Added 0.5ul nuclease and 1ul enhancer to each tube. Cut at 42deg for 45 mins. Ran on gel: