Alice:LabNotes/2008-8-13
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comparison of PCR result using different Ribo concentration[edit]
reaction system 1: x48 H2O 80.1ul 3844.8ul 10x Stoffel buffer 10ul 480ul MgCl2(25mM) 6ul 288ul dNTP(10mM each, NEB) 2ul 96ul Primer (APV6) 0.4ul 19.2ul primer (Ribo V6 100uM) 0.4ul 19.2ul 50x SYBG I 0.4ul 19.2ul template(CES22K #4) 0.1ul 4.8ul Jumpstart Taq 0.6ul 28.8ul Reaction system 2: H2O 80.7ul 10x Stoffel buffer 10ul MgCl2(25mM) 6ul dNTP(10mM each, NEB) 2ul primer (Ribo V6 100uM) 0.2ul primer (APV6) 0.2ul 50x SYBG I 0.4ul template(CES22K #4) 0.1ul Jumpstart Taq 0.6ul 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 15C hold
PCR curve: TBE gel Result: File:8-13-08.jpg File:2008-08-13 22K with ribo V6.jpg Note:error on above image, the ladder should be 25bp instead of 10bp.
22K probe preparation[edit]
reaction system 1: x48 H2O 80.5ul 3864ul 10x Stoffel buffer 10ul 480ul MgCl2(25mM) 6ul 288ul dNTP(10mM each, NEB) 2ul 96ul primer mix (Ap6) 0.4ul 19.2ul 50x SYBG I 0.4ul 19.2ul template(CES22K #4) 0.1ul 4.8ul Jumpstart Taq 0.6ul 28.8ul reaction system 2: x48 H2O 80.1ul 3844.8ul 10x Stoffel buffer 10ul 480ul MgCl2(25mM) 6ul 288ul dNTP(10mM each, NEB) 2ul 96ul Primer (APV6) 0.4ul 19.2ul primer (Ribo V6 100uM) 0.4ul 19.2ul 50x SYBG I 0.4ul 19.2ul template(CES22K #4) 0.1ul 4.8ul Jumpstart Taq 0.6ul 28.8ul 94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min -> 15C hold
Step 2: Probe purification with 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 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 250 uL dH2O. Transfer the liquid to two 0.2ml tube.
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 2 hours followed by 90C for 10 min, and leave it at 4C forever. File:22K with ribo V6 and APv6 after exonuclease 8-19-08.jpg
Step 4: Purify the reaction with Qiaquick columns 1. for each 150ul tube, add 750 uL of PBI buffer, mix well, load 450 ul each time to column (twice with total of 900 uL) 2. Spin at top speed (14000 rpm) for 1 min 3. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column 4. Spin at top speed for 1 min 5. Empty the collection tube, spin at top speed for 1 min 6. Carefully transfer the columns to clean 1.6 mL tubes 7. Place the column/tube assemblies to 37C incubator, incubate for 5 min 8. Add 45 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min. 9. Pool DNAs from the two tubes into one, measure the concentration with Nanodrop.
NanoDrop result: reaction system using Ribo V6 primer: tube 1: 55.9 ng/ul tube 2: 55.7 ng/ul reaction system using APV6 primer: tube 1: 61 ng/ul tube 2: 64 ng/ul
Step 5: Incubation with USER enzyme: add 6 uL of 10X DpnII reaction buffer and 10 uL of USER (1K/ml)enzyme and last 8 hour on 37C. Take 4ul out to run gel later.
Step 6: Annealing of DpnII oligo and incubation with DpnII. 1. Add 1uL of 10X DpnII reaction buffer and 10 uL of 100 uM RE_RE_DpnII_n2S guide oligo. Denature at 95C in PCR machine for 10 min. Turn off the machine and wait for 20 min. 2. Wait until th machine cool to 37C then add 2 uL of DpnII restriction enzyme (50K/ml) to reaction, incubate for about 4 hours at 37C.
*for reaction system using riboV6, anneal with DpnII oligo first, then add 1N KOH to make the final concentration 200mM, incubate at 85C for 30min, then add the same volume of 1N HCl to neutralize the solution. After neutralization, remove the salt by using G25 columns. Then purify the product using MinElute columns.* Added 65 ul of 400mM KOH to 65ul of product(riboV6), incubate at 85C for 30 min. Then added 26ul of 1N HCl to neutralize the solution. Purify the product with MinElute columns and measure the concentration using Nanodrop. File:2008-08-28 22K after DpnII.jpg File:8-29-08 riboV6 after KOH and G25.jpg TBU gel of standard and RiboV6 comparison TBU gel of RiboV6 after KOH and G25 column 8-29-08
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 8 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. Results: 2D gel of the RiboV6 (8-29-08) File:ZhangLab 2 2008-08-29 15hr 33min.jpg
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 4uL of 5ng/uL 90-mer, 4ul of H2O 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. 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. %yield for standard probe is 6ng/ul, and the bands for RNA based (RiboV6) probe sample is too weak to be quantified. File:9-2-08 22K quantification.jpg Result: RiboV6 can be a good substitute for this protocol, and the above gel results shows that everything works fine until after DpnII. The protocol still needs some improvement after DpnII, maybe increase increase the temperature or the digestion time to increase the %yield.