AlanFung:Protocol/Probe Preparation
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Probe Preparation
Samples & Materials
- Jan_09_CPG_D40809 100K Oligo (10uM)
- EconoTaq Plus 2X Master Mix Lucigen Cat. 30035-1
- AP1V61U 5' - G*G*G TCA TAT CGG TCA CTG T/3deoxyU/ - 3'
- AP2V6 5' - /5Phos/CAC GGG TAG TGT GTA TCC TG - 3'
- AP1V61U AP2V6 Primer Mix (50uM)
- Nuclease-free Water (not DEPC-treated) Ambion Cat. AM9937
- 50X SYBG I
- 3M NaOAC
- GlycoBlue
- 100% Ethanol
- 75% Ethanol
- QIAquick PCR Purification Kit Qiagen Cat.28104 & 28106
- Buffer PB Qiagen Cat. 19066
- QIAquick Spin Column
- Buffer PE Qiagen Cat. 19065
- Nanodrop
- 10X EXO Buffer
- Lambda Exonuclease
- USER Enzyme NEB Cat. M5505L
- DpnII NEB Cat. R0543M
- 10X DpnII buffer [[NEB Cat.
- RE-DpnIIs guide oligo
- 2D Gel
- 10X TBE buffer
- 0.5X TBE buffer
- orbital shaker
- 10bp Ladder
- SYBR Gold
Overview
- PCR reaction system setup
- Probe purification with ethanol precipitation
- Purify the reaction with Qiaquick columns
- Adapter Removal
- Page Denaturing Gel
- Purify the product from the gel
- Precipitation
- Quantification of DNA using denaturing gel and a quantitative ladder
Procedures
Step 1: PCR reaction system setup
- Thaw Econo Taq on ice
- Prepare strips and tubes on cooling rack
- Add in Content in table to tubes accordinly
Content | Volume | X24 | X48 | X72 | X96 | 1.5 mL Tube |
Jan_09_CPG_D40809 (10uM) | 0.2uL | 4.8uL | 9.6uL | 14.4uL | 19.2uL | 2.4uL |
Econo Taq | 50uL | 1250uL | 2400uL | 3600uL | 4800uL | 650uL |
AP1V6 AP2V6 Mix (50uM) | 0.4uL | 9.6uL | 19.2uL | 28.8uL | 38.4uL | 4.8uL |
RNAse free H2O | 50uL | 1200uL | 2400uL | 3600uL | 4800uL | 600uL |
50X SYBG I | 0.4uL | 9.6uL | 19.2uL | 28.8uL | 38.4uL | 4.8uL |
Total Volume | 101uL | 2424uL | 4848uL | 7272uL | 9696uL | 1212uL |
- Mix content in 1.5 mL tube vortex and spin down in the order of water>SYBR Green>Primer >template>econotaq
- Aliquot 105uL using P200 pipette to strip tubes on cooling rack
- Seal strips with strip caps
- Place into Real Time PCR
- Perform
94C 2M > (94C 30S > 58C 30S > 72C 30S>Plate Read) X16 > 72C 3M > 15C Hold
Step 2: Probe purification with ethanol precipitation
- Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content | Volume Needed | Volume |
100% Ethanol | 2.5X Sample Volume | 6000uL |
3M NaOAC | 0.1X Sample Volume | 240uL |
Glycoblue | 1/300X Sample Volume | 8uL |
Sample | Sample Volume | 2400uL |
Total Volume | 8648uL |
- Mix all content by repeat pipetting using multichannel pipette
- Pool all 24 wells PCR product into tray using multichannel pipette(P300)
- Mix all content by repeat pipetting using multichannel pipette
- Aliquot about 3750uL to 2 x 15mL tube
- Store in -80C for more than 20 mins or forever
- Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
- Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C
- Remove all liquid CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE
- Add in 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 1.5mL tube
- Centrifuge at 10,000rpm for 5min at 4C
- Remove all liquid, air dry sample for 5min or until samples dried out
- Resuspend the DNA with 100uL RNAse free H2O
Step 3: Purify the reaction with Qiaquick columns
- Add 5X sample volume of PB buffer into each tube
- Mix by repeat pipetting
- Load mixture onto column
- Centrifuge at 14,000rpm for 1min
- Discard flow through and add 750uL PE buffer to each column
- Repeat Centrifuge
- Discard flow through
- Repeat centrifuge
- Transfer columns to clean 1.5mL tubes
- Place the column/tube assemblies on bench top wait for 2min
- Add in 50uL EB Buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5min
- Repeat centrifuge
- Measure concentration with Nanodrop
Step 4: Adapter Removal
Exonuclease treatment
- Add in 3uL Lambda Exonuclease (5K/mL) for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube
- Add in (1/10 of total volume) 6uL 10X Exo buffer
- Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR
Purify the reaction with Quaquick columns
- Add 5X sample volume of PB buffer into each tube, mix well
- Load mixture onto column
- Centrifuge at 14,000rpm for 1min
- Discard flow through and add 750uL PE buffer to each column
- Repeat Centrifuge
- Discard flow through
- Repeat centrifuge
- Transfer columns to clean 1.5mL tubes
- Place the column/tube assemblies on bench top wait for 2min
- Add in 50uL EB buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
- Repeat centrifuge
- Measure concentration with Nanodrop
Incubation with USER enzyme:
- Add 5ul DpNII 10x buffer and 2 uL of USER (1K/ml)enzyme incubate for 2 hours on 37C.
Annealing of DpnII oligo and incubation with DpnII.
- add 6ul of 10X DpnII buffer and 2uL RE-DpnIIs guide oligo (100uM)
- Perform 94C 5m > 60C 5m > 0.1C/sec to 37C > add 4ul of DpnII restriction enzyme -> 37C 2 hours > 4C hold
- Qiaquick column purification
- Elute with 60uL EB Buffer
Step 5: Page Denaturing Gel
- Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel
- Load 2 gels into the gel running module
- Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
- Pre-run the gel for 30 min at 200V
- Dilute sample to no more than 40ng/uL
- Dilute TBE-Urea sample buffer (2X) to 1X in each tube.
For 2 gels; Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer
- Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins
- Flush the wells very well with P1000 & P200 pipette to remove all urea.
NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP
- Load the samples into the wells, run at 200V for 30 min.
- Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.
- Place tray on an orbital shaker for ~5 min.
Step 6: Purify the product from the gel
- 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. Use two assemblies for each PAGE gel.
- 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.
- Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
- Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
- Spin down all tubes at 13,000rpm for 2min
- In the PCR hood, transfer the liquid layer of 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 7: Precipitation
Turn on centrifuge let it cool down to 4C
- Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
- Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
- Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
- Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
- Let dry in hood ~ 10 min.
- Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Store at 4C if needed, spin down before proceeding
Step 8: Quantification of DNA using denaturing gel and a quantitative ladder
Combine all tubes into a 1.5mL tube
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 buffer 3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, 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.