Alice:LabNotes/2008-4-10

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padlock probe library preparation[edit]

Step 5: Incubation with USER enzyme:

 1. Divide the 150ul probe sol into two tubes. In each tube, add 10uL of 10X DpnII reaction buffer and 5 uL of USER (1K/ml)enzyme and last 2 hour on 37C. 
 Take 4ul out to run gel later.

Step 6: Annealing of DpnII oligo and incubation with DpnII (each tube).

 1. Add 5 uL of 100 uM DpnII guide oligo (SNP and CpG probe need different 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 (the temperature cools from 95C to around 40C).
 4. Add 10 uL of DpnII restriction enzyme (10K/ml) to reaction, incubate for about 2 hours at 37C, take 4ul out to run the gel.
 5. add 5 uL of USER (1K/ml)enzyme and last another 12 hour. take 4ul out to run the gel.


File:2008-04-10 Alice 27K probe preparation.jpeg

From left to right is : Product from step 6, product from step 4, ladder.