Alice:LabNotes/2008-1-28
Experiment 1: Repeat Padlock preparation continued
Step 2: Probe purification with ethanol precipitation
1. pool all the 16 reactions into one 10 mL tube, add 160 uL 3M NaOAC, 5 uL GlycoBlue, 3.2 mL 100% Ethanol.
2. Store at -70C for 26 min.
3. Spin at 4000rpm for 15 min at 4C.
4. Remove all liqid, you should see a blue pallette at the bottom of the tube.
5. Add 750 uL cold 75% Ethanol, piette well and transfer to a 1.6 mL tube.
6.Spin at 10000 rpm for 5 min at 4C.
7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 82 uL dH2O.
Step 3: Exonuclease treatment
1. Add 10 uL 10X Exo buffer and 8 uL Exo for total volume of 100uL.
2. Incubate at 37C for 2 hours followed by 75C for 15 min, and leave it at 4C forever.
Step 4: Cleavage reaction with USER enzyme
1. Add 8uL of USER enzyme into reaction.
2. Incubate at 37C for 1 hour.