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

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05/15/2015 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