Haynes Lab:Notebook/CRISPR Editing/2015/05/15

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Ran nested PCR reactions for dilution experiment and KAH228, GFP, g034 experiment done yesterday on a gel. All had correct bands and both the blanks were negative. Purified them using the Qiagen Qiaquick PCR purification kit.

Cloning GFP behind Cas9
Resuspended 200ng of dried gblock with 10ul water for a final concentration of 20ng/ul
Cut 1ug of pX330 and 40ng of gblock

37°C for 30min
65°C for 20min
thing '
backbone 1ug
10x Cutsmart buffer 5ul
FseI 1ul
Water to 50ul

thing '
gblock insert 40ng
10x Cutsmart buffer 2.5ul
FseI 1ul
Water to 25ul

Ran 2ul backbone on gel to confirm FseI activity. Saw one band at correct size for backbone, conclude FseI completely cut.
Added 1ul of Thermo fastdigest EcoRI to each reaction

37°C for 30min
85°C for 5min

Put at -20°C