Alice:LabNotes/2008-1-17

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Revision as of 23:58, 18 January 2008 by KunZhang (talk | contribs) (New page: <b>Experiment 1: Padlock preparation continued</b><br> Step 6: Annealing of DpnII oligo<br> 1. Add 15uL of 10X DpnII reaction buffer and 5 uL of 100 uM DpnII guide oligo.<br> 2. Denature a...)
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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.