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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 =October 28, 2009= ==Step 6: Annealing of DpnII Oligo & Incubation with DpnII == (Optional) Remove 1 ul for PAGE 1. Prepare reaction (1x, 78) ** from USER, 72 ul ** DpnII buffer 10x, 1 ul ** DpnII-V4 guide oligo (100uM), 5 ul 2. Program ("DpnII") ** 95C - 10 min ** Gradient 0.1C/s to 60C ** 60C - 20 min ** 37C - 4 hours ***At 1-2 into this step, add 2ul DpnII restriction enzyme ** 4C - forever 3. Leave overnight at 4C =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 =October 30, 2009= ==Step 10: Quantification of DNA using Denaturing gel & a quantitative ladder== * Combine DNA from both tubes. 1. Prepare samples: ** Low mass DNA ladder, 3ul, dH20, 6ul, TBE urea buffer (2x), 9ul (18ul totally) ** DNA sample, 1ul, dH20, 9 ul, TBE urea buffer (2x), 10 ul (20 ul totally) 2. Pre-run gel, 6% TBU gel, 10-12 wells, 200V->10min 3. Denature DNA + loading dye using PCR machine at 75C -> 8min 4. Transfer to ice quickly to avoid denaturation 5. Flush wells to remove urea 6. Load samples: ** DNA: 10 ul, 5.0 ul, 2.5 ul ** Ladder: 6 ul, 6 ul, 3 ul, 3 ul 7. Run at 200V -> 30min 8. Stain gel w/ 5ul SYBR gold in 100ml 0.5xTBE 9. Place on orbital shaker for 4 min 10. Quantify gel to calculate yield: [[Image:Dinh-ZhangLab_2_2009-10-30_gel_quantification_20ul_CpG_30K.jpg|200px|thumb|left|PAGE Quantification = 12ng/ulx20ul]]
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