Dinh:Probes Prep
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Padlock probes preparation[edit]
Production PCR[edit]
- 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[edit]
- 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 at least 1 hour.
- 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. (Optional: dry residual ethanol by warming columns in thermomixer for 5 minutes at 50C). Elute each with 30 uL of pre-warmed EB.
- Nanodrop to quantify yield.
Removal of amplification adapters[edit]
- NOTE: Make sure Lambda exo reaction is complete (check with TBE gel, should see 1 ssDNA band, not two) & use fresh DpnII buffer & use DpnII at 50 units/uL to get the best digestion efficiency.
- Lambda Exonuclease reaction:
Lambda exo buffer (10x) : 15 uL Lambda exo (5 units/uL) : 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 (1 unit/uL, 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)
- 1 unit of USER digests 10 pmol of uracils at 37C for 15 minutes.
- Incubate reaction at 37C for at least 1 hour.
- Prepare DpnII oligo mix:
RE-DpnII_2N V4/6 guide oligo : 12 uL DpnII buffer (10x) : 2 uL (Keep DpnII buffer on ice or frozen when storing, do not use very old DpnII buffer) H2O : 5 uL
- Add 19 uL of DpnII oligo mix to each USER reaction mix.
- Program:
94C for 5 minutes -> 60C for 20 min, ramp -0.1C/s to 37C -> 37C for 3 minutes
- Add 1 uL of DpnII (50 units/uL) while tubes are at 37C
- Incubate at 37C for 2 hours.
- Add more DpnII (important if >= 5 ug of ssDNA was used):
DpnII (NEB, 50 units/uL) : 1 uL DpnII buffer (10x) : 1 uL H2O : 8 uL
- Add 10 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. (Optional: dry residual ethanol by warming columns in thermomixer for 5 minutes at 50C). Elute with 30 uL of pre-warmed EB.
PAGE size selection[edit]
- 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 40 minutes at 200 V. (Run lower volt to get better band resolution).
- Add 1.5 mL TE buffer to sheared gel.
- Rotate overnight at room temperature.
- 50C for 15 minutes at 900 rpm on thermomixer. (Or 37C for 1 hour at 900 rpm). Yield with 37C/1 hour seems decent, not sure which is better.
- Quickly spin-down tubes & filter through 1-2 Nanosep columns. Keep tube warm.
- 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 at least 1 hour.
- 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 pre-warmed nuclease-free water.
- QUBIT ssDNA to quantify yield.
Agarose gel size selection[edit]
- Prepared >4% agarose gel (Apex multipurpose agarose). 1 g in 25 mL. UV for 15 minutes.
- Put on heater/mixer at 200 degC let sit for about 5-10 minutes. Turn off the heat.
- Fill a 2 L beaker partly with water and put the flast with melted agarose inside. Continue stirring for 10 minutes to cool the agarose solution.
- Added 2uL of Sybr Gold per 25mL of agarose solution when the agarose solution have cooled enough to hold for 15 seconds.
- Cast a gel of about 7 mm thickness with 3-5 mm wells.
- Load at least 700 ng into each well. Purification will not work with less than 700 ng minimum input.
- Ran at 135V for 45 minutes, the probes were in the bottom quarter of the gel by the end of electrophesis run.
- Gel image:
- After sample finished running. Weight a 50 mL tube.
- Cut out 2 wells per tube and re-weigh to calculate 6X volume of QX1 buffer to add to each tube.
- Add 10 uL of QIAX suspension solution to each tube.
- Incubate at 60 degC for 6 minutes. Vortex every 2 minutes.
- Label 2 mL tubes for spinning down the solution. Remove supernatant after each spin.
- Wash with 500 uL QX buffer once.
- Wash with 500 uL PE buffer three times.
- Dry the pellet in the vacuum for 2 minutes.
- Resuspend the pellet with water.