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