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==probe preparation== Step 1: Real time PCR reaction system x12 x8 H2O 70.5ul 846ul 6768ul 10x hotstart Taq buffer 10ul 120ul 960ul MgCl2(25mM) 6ul 72ul 576ul dNTP(10mM;NBE) 2ul 24ul 192ul Ap1V4IU(100uM) 0.2ul 2.4ul 19.2ul Ap2V4(100uM) 0.2ul 2.4ul 19.2ul 50x SYBG I 0.4ul 4.8ul 38.4ul template(20nM) 0.1ul 1.2ul 9.6ul hotstart Taq 0.6ul 7.2ul 57.6ul Total 100ul 1200ul 9600ul Reaction program: 94c 2min -> (94c 30sec -> 60C 1.5min -> 72C 1min)x 20~22 cycles -> 72C 5min -> 15C hold. step 2: ethanol precipitation 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 25 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. Step 3: Exonuclease treatment 1. Add 15 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5K/ml) for total volume of 150uL(each tube). 2. Incubate at 37C for 1 hour 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 each 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 45 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min. measure the concentration with Nanodrop. Step 5: Incubation with USER enzyme: Add 6ul DpNII 10x buffer and 8 uL of USER (1K/ml)enzyme and last 2 hour on 37C. Step 6: Annealing of DpnII oligo and incubation with DpnII. 1. Add 1uL of 10X DpnII reaction buffer and 10 uL of 100 uM DpnII-V4 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 4 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. <BR> 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<BR> 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,<BR> 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),<BR> 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 <BR> 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. *I transfer 500ul 75% ethanol and DNA into another tube and another to accumulate the probe. Step 10: Quantification of DNA using denaturing gel and a quantitative ladder 1. Add 3uL of low mess ladder and 6ul H2O and 9 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. <BR> Load the ladder as 3, 3, 6, 6 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. <BR> Then quantify the gel to calculate the % yield.
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