Alice:LabNotes/2008-1-17
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Experiment 1: Padlock preparation continued
Step 6: Annealing of DpnII oligo
1. Add 15uL of 10X DpnII reaction buffer and 5 uL of 100 uM DpnII guide oligo.
2. Denature at 95C in PCR machine for 10 min.
3.Turn off the PCR program and let samples cool off in the block for ~15 min.
Step 7: Incubation with DpnII restriction enzyme
1. Add 10 uL of DpnII restriction enzyme to reaction.
2. Incubate for about 3 hours at 37C, last one hour add 5 uL of USER enzyme to increase efficiency.