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==cpgJan09 preparation== PCR reaction x 48 x96 H2O 49ul 2352ul 4704 EconoTaq 2xMastermix 50ul 2400ul 4800 Ap1V6IU (100uM) 0.3ul 14.4ul 28.8 Ap2V6 (100uM) 0.3ul 14.4ul 28.8 SYBR green (50x) 0.4ul 19.2ul 38.4 template(cpgJan09A,13nM) 0.05ul 2.4ul 4.8 Total 100ul 4800ul 9600 *Left 2 wells for CPG09B (Prepared by Ida) Reaction program: 94c 2min -> (94c 30sec -> 60C 1min -> 72C 45sec)x 19ycles -> 72C 5min -> 15C hold. *Repeat for the 2nd plate in regular PCR step 2: ethanol precipitation *Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray {| border="1" align="center" | align="center" style="background:#f0f0f0;"|'''Content''' | align="center" style="background:#f0f0f0;"|'''Volume Needed''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- |100% Ethanol||align="right"|2.5X Sample Volume||align="right"|6000uL |- |3M NaOAC||align="right"|0.1X Sample Volume||align="right"|240uL |- |Glycoblue||align="right"|1/300X Sample Volume||align="right"|8uL |- |Sample||align="right"|Sample Volume||align="right"|2400uL |- |Total Volume |colspan="2" align="right"|8648uL |- |} 1. pool the 24 reactions into one 16 mL tube, add 240 uL 3M NaOAC, 8 uL GlycoBlue, 6 mL 100% Ethanol. 2. Store at -70C for more than 20 min. 3. Spin at 4000rpm for 15 min at 4C. 4. Remove all liqid, you should see a blue pallette at the bottom of the tube. 5. Add 750 uL cold 75% Ethanol, piette well and transfer to a 1.6 mL tube. 6. Spin at 10000 rpm for 5 min at 4C. 7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 125 uL dH2O. Transfer the liquid to 0.2ml tube. Step 3: Exonuclease treatment 1. Add 15 uL 10X Exo buffer and 5 uL Lambdo Exonuclease (5K/ml) for total volume of 150uL(each tube). 2. Incubate at 37C for 1 hours followed by 90C for 10 min, and leave it at 4C forever. Step 4: Purify the reaction with Quaquick columns 1. for each tube, add 5x PBI buffer, mix well, load to two column. 2. Spin at top speed (14000 rpm) for 1 min. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column.Spin at top speed for 1 min 3. Empty the collection tube, spin at top speed for 1 min, transfer the columns to clean 1.6 mL tubes 4. Place the column/tube assemblies to roomtemprature for several mins. 5. Add 30 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min. measure the concentration with Nanodrop. Nanodrop results: 106.5ng/ul x 60ul x4 = 25ug; 80.9ng/ul x 60 x 4; Step 5: Incubation with USER enzyme: Add 8ul DpNII 10x buffer and 6 uL of USER (1K/ml)enzyme and last 8 hour on 37C. Step 6: Annealing of DpnII oligo and incubation with DpnII. 1. Add 1uL of 10X DpnII reaction buffer and 5 uL of 100 uM DpnII-V6 guide oligo. Denature at 95C in PCR machine for 10 min. 2. cool 0.1C/sec to 60C and hold 60C for 20mins -> 37C 1min -> Add 2 uL of DpnII restriction enzyme (50K/ml) to reaction, incubate for about 2 hours at 37C. Step 7: Page Denaturing Gel 1. Add TBE-Urea sample buffer (2x) in each tube. 2. Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer. 3. Denature at 75C for 15 min, then quickly transfer to ice, wait for 1 min. 4. Use a 2D well denaturing gel, add 0.5x TBE buffer. Flush the wells very well to remove all urea. 6. Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min. 7. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min. Step 8: Purify the product from the gel 1. Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. You need two assemblies for each PAGE gel. 2.Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly. 3. Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. 4. Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 50C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. 5. In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube. Step 9: Precipitation 1. Add 0.1x of 3M sodium acetate and 2.5x 100% ethanol. 2. Add 1uL Glycoblue so that one can precipitate better and see the DNA. 3. Freeze in -70C for 20 min, spin at 14,000 rpm for 15 min at 4C, discard supernatant with pipette. 4. Add 500 uL of 75% ethanol to wash DNA, spin at 8000 rpm for 5 min at 4C, discard supernatant with first large tip and then small tip to remove as much liquid as possible. 5. Let dry in hood ~ 10 min. 6. Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min. Step 10: Quantification of DNA using denaturing gel and a quantitative ladder 1. Add 8uL of 2ng/uL 90-mer and 8 uL of 2x TBE-Urea buffer 2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE-Urea buffer. 3. Denature the DNA with loading dye on thermalcycler 75C for 15 min, then quickly transfer to ice, wait for 1 min. Note: Transfer to ice to keep the samples from further denaturation. 4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. 5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min. Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL. 6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.
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