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Dinh:Protocols/Probes Prep Oct27
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=October 27, 2009= ==Step 1: Real Time PCR Probe Amplification== 1. Prepare 96 Reactions (12x or 1.2ml per tube): * 1. ddH20, 600 ul * 2. EconoTag 2x Master Mix, 600 ul * 3. AP1V4IU (100uM), 3.6 ul * 4. AP2V4 (100uM), 3.6 ul * 5. SYBR Green (50x), 4.8 ul * 6. Template CpG30k (20nM), 0.6 ul 2. Program ("1 step probe preparation"): * 1. 94C - 2 min * 2. 94C - 30 s * 3. 60C - 1 min * 4. 72C - 45 s * 5. Plate Read * 6. Cycle to 2 for 22 more times * 7. 72C - 5 min * 8. 15C - forever * 9. end 3. Stopped at 22 cycles. ==Step 2: Ethanol Precipitation== 1. In Solution Basin (55ml) Add: ** NaOAc (3M), 960 ul ** GlycoBlue, 32ul ** Ethanol 100%, 24ml 2. Pool all reactions into Solution Basin (55ml) 3. 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, 139ul): ** PCR product, 112 ul (measured) ** 10x Exo Buffer, 14 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. 67.12 ng/ul 480 ul total volume => 32.2 ug ==Step 5: Incubation with USER Enzyme == (Optional) Remove 1 ul for PAGE 1. Prepare reaction (1x, 72 ul) ** Probes, 60 ul ** DpnII 10x Buffer, 8 ul ** USER (1000 U/ml), 6 ul 2. Program ** 37C -> 3 hours
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