Haynes Lab:Notebook/CRISPR Editing/2016/02/27

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02/27/2016

Finally found the FseI in the -80! Cut 1ug of pX330A_dCas9_1x4 vector from addgene and 80ng insert (gB009) with 1ul FseI for 1h at 37degC then deactivated at 65degC for 20min. Did three tubes of the backbone.

1ug bb
5ul cutsmart
1ul FseI
40.41ul H2O



I didn't like that there were extra bands in the purified cut MV13-KAH60. Decide to redigest with less enzyme for shorter time. Cut 2ug of MV13-KAH60 vector with 0.3ul SgrAI for 18 min at 37degC then deactivated at 65degC for 20min. Did two tubes of the backbone.

2ug bb
5ul cutsmart
0.3ul SgrAI
38ul H2O



Ran all cut backbones on gel, all look good!
Screenshot 2016-03-12 20.30.05.png

Added 1ul of XbaI to MV13-KAH60 cut with SgrAI, inc at 37degC for 18 mins. Column purified, another ugly spec, maybe it's the kit? Got 48ng/ul.

Column purified gB014 PCR product with P169/216. Got 132ng/ul but the peak was ugly. Cut 2ug total, two reactions for 15 mins:

8ul insert (1ug)
1ul BbsI
1ul SpeI
2ul FD buffer
8ul water

Column purified

Cut pX330A_dCas9_1x4 and gB009 with EcoRI for 15 min, just added 1ul to each tube. PCR purified the backbone, heat inactivated the insert at 80degC for 5mins. Cooled slowly in PCR machine using oligo annealing protocol.