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Dinh:Protocols/Probes Prep Nov17
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=November 18, 2009= ==Step 2: Ethanol Precipitation== 1. In Solution Basin (55ml) Add: NaOAc (3M), 960 ul GlycoBlue, 32ul Ethanol 100%, 24ml 2. Pool all reactions from 1 plate into Solution Basin (55ml) 3. For each plate, transfer equally to 8x15 ml tube. Store at -70C for more than 20 min 4. Turn on centrifuge (might need to switch the "swirlers") 5. Spin at 4000rpm->20min->4C 6. Remove all supernatant, leave the blue pellet 7. Wash with cold ethanol (75%) ~750ul 8. Move pellet to 1.5ml tube, vortex/mix well 9. Spin at 10,000rpm->5min->4C 10. Remove all supernatant, leave 5 min to air dry. 11. Resuspend DNA with 125 ul dH20 12. Leave in 37C incubator for 30 min. Vortex to fully dissolve DNA. 13. Pool the content of 8 tubes into one. ==Step 3: Exonuclease Treatment== 1. Prepare reaction (1x, 150ul): ** PCR product, 130 ul (measured) ** 10x Exo Buffer, 15 ul ** Lambdo Exonuclease (5000 U/ml), 5ul 2. Program ("Exo") ** 37C - 1 hr ** 90C - 10 min ** 4C - forever ==Step 4: Purification with QuaQuick Columns== 1. Add 5x PBI buffer (750 ul) 2. Mix well 3. Load equally to 2 columns 4. Spin at 14000 rpm->1min 5. Empty collection tubes 6. Add 750 ul wash buffer (PE) 7. Spin at 14000rpm->1min 8. Empty collection tubes & spin again for 2 min 9. Air dry for 5 min 10. Transfer to labeled 1.5ml tubes 11. Add 34 ul ddH20 to each center of each column 12. Wait 1 min 13. Spin at 14000rpm->1 min ==NANODROP== A. 100 ng/ul 720ul total volume => 72.0 ug ==Step 5: Incubation with USER Enzyme == 1. Prepare reaction (1x, 74 ul) ** Probes, 60 ul ** DpnII 10x Buffer, 8 ul ** USER (1000 U/ml), 6 ul 2. Program ** 37C -> 3 hours
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