Dinh:Probes Prep
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Padlock probes preparation
Production PCR
- I usually prepare 20x 100 uL reactions in a 2.0 mL tube. (For 96 wells plate, prepare 100 reactions & aliquot 104 uL to each).
- qPCR Reaction:
KAPA Master Mix(2x) : 1000 uL H2O : 980 uL AP1V4/6(100uM) : 8 uL AP2V4/6(100uM) : 8 uL Template (10nM) : 4 uL
- Program:
95C for 30s -> [95C for 10 s -> 55C for 20s -> 72C for 30s] x 13-15 cycles -> 72C for 2 minutes
Ethanol precipitation
- Pool 20 reactions into 2x 15 mL tube
- Add 2.5x 100% ethanol, 0.1x 3M sodium acetate, and 8 uL of glycoblue
- Store in -80C for overnight (or 40 minutes)
- Spin at >4000 rpm for 40 minutes at 4C
- Remove supernatant & add 1 mL 80% ice-cold ethanol
- Spin at >4000 rpm for 15 minutes at 4C
- Remove supernatant & air dry for 10 minutes
- Add 150 uL of water to each tube & pool together.
- Purify with 3x Qiaquick PCR cleanup columns. Elute each with 30 uL of EB
- Nanodrop to quantify yield.
Removal of amplification adapters
- Lambda Exonuclease reaction:
Lambda exo buffer (10x) : 15 uL Lambda exo : 10 uL dsDNA probes (up to ~20 ug) : 125 uL
- Incubate reaction at 37C for 1 hour.
- Purify with 2x ssDNA/RNA cleanup columns. Elute each with 30 uL of EB.
- Nanodrop to quantify yield.
- USER digestion reaction:
USER (NEB) : 8 uL DpnII buffer (10x) : 10 uL ssDNA probes (up to 5 ug) : 82 uL (100 ng/uL is 2.02 pmol/uL, 150 nt oligos)
- Incubate reaction at 37C for at least 1 hour.
- Prepare DpnII oligo mix:
RE-DpnII V4/6 guide oligo : 5 uL DpnII buffer (10x) : 2 uL (Keep DpnII buffer on ice or frozen when storing, do not use very old DpnII buffer) H2O : 11 uL
- Add 18 uL of DpnII oligo mix to each USER reaction mix.
- Program:
94C for 2 minutes -> 60C for 30s, ramp -0.1C/s to 37C -> 37C for 3 minutes
- Add 2 uL of DpnII (100 units) while tubes are at 37C
- Incubate at 37C for 2 hours.
- Add more USER (important if >= 5 ug of ssDNA was used):
USER (NEB) : 3 uL DpnII buffer (10x) : 2 uL H2O : 15 uL
- Add 20 uL to each reaction.
- Incubate at 37C for one hour. During the second USER digestion, ~5 uL of the digestion reaction can be checked for digestion completion.
- Purify with 1x ssDNA/RNA cleanup column for each reaction tube. Elute with 30 uL of EB.
PAGE size selection
- UV all gel running buffer and chamber.
- UV shearing apparatus.
- Prepare 3x (2.0 mL tube + 0.5 mL tube) per gel. Cut off the caps of 2.0 mL tube to enable centrifuge at 15,000 rpm.
- Puncture 0.5 mL gel with 20 gauge needle.
- Run 6% gel for 30 minutes at 200 V.
- Add 1.5 mL TE buffer to sheared gel.
- Rotate overnight at room temperature.
- 50C for 15 minutes on thermomixer.
- Quickly spin-down tubes & filter through 1-2 Nanosep columns.
- Pool flow-through in 1x 15 mL tube.
- Add 2.5x 100% ethanol, 0.1x 3M sodium acetate, and 8 uL of glycoblue
- Store in -80C for overnight (or 40 minutes)
- Spin at >4000 rpm for 40 minutes at 4C
- Remove supernatant & add 1 mL 80% ice-cold ethanol. Transfer to 1x 1.5 mL tube.
- Spin at >10,000 rpm for 15 minutes at 4C
- Remove supernatant & vacumm dry for 10-30 minutes
- Add 30 uL of water.
- QUBIT ssDNA to quantify yield.