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=October 29, 2009= ==Step 7: PAGE Denaturing Gel== * Use "Probe Prep Only" gel box * Use PCR tubes * Load 2ug/2D well 1. Prepare samples: * 1 DpnII reaction mix: 76 ul, TBE Urea Sample Buffer (2x), 76 ul. (152ul) * Invitrogen 10bp ladder, 0.5ul, dH20, 4.5 ul, TBE Urea Sample Buffer (2x), 5ul.(10ul) * Loaded 76 ul sample+buffer mix per well * Loaded 5 ul ladder+buffer mix per well 2. Denature at 75C -> 8 min 3. Transfer to ice quickly. 4. Pre-run gel, 2D well, TBU gel, 200V-> 10min 5. Flush wells to remove all urea 6. Load samples quickly (hold tubes so that you are not indirectly touching the solution inside). 7. Run at 200V->30min 8. Stain with SYBR Gold (5ul in 100ml 0.5xTBE) in a clean tray. 9. Place on orbital shaker for 1-2 min. ==Step 8: Purify the product from the gel == * Switch gloves when using PCR hood 1. Prepare gel-shearing assemblies (2x) using non-stick 0.5ml and 1.6ml tubes. 2. Use G22 needle to punch a hole at the bottom of the 0.5 ml tube 3. Cut out 100-110 nt band. Divide into 2 equal pieces. Chop up each and place pieces into a gel-shearing assembly. 4. Centrifuge at 14000 rpm for 3 min to shear gel. Move any remaining pieces to bigger tube. Remove the 0.5 ml tube 5. Add 400ul clean 1x TE buffer. 6. Vortex at 37 C for 1 hour 7. In PCR hood, transfer gel mix to Nanosep columns 8. Spin at top speed 15000 rpm for 3min 9. Transfer supernatant to 1.5 ml tube (non-stick). ==Step 9: Ethanol Precipitation== 1. Prepare reaction: ** Pool the content of 8 Nanosep flowthrough to 1x15ml tube. = total 16x15 ml tubes **NaOAc (3M), 0.1xsample vol **100% ethanol, 2.5xsample vol **Glycoblue, 1/300thxsample vol * Make sure that tubes are within 0.1g of each other by adding 100% EtOH (in increments of 50ul). 2. Freeze at -70C for 20 min 3. Spin at 4000rpm->25min->4C 4. Remove supernatant 5. Wash with 500 ul 75% ethanol. Pool contents of 8 tubes into 1x1.5ml tube. 6. Spin at 8000rpm->5min->4C 7. Remove all supernatant with pipettors 8. Leave in hood to air dry ~10 min 9. Add 10ul ddH20 to each tube to resolve DNA 10. Leave at least 15 min, then leave overnight at 4C
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