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 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

  • 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 50units/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 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 (50 units/uL) 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. (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

  • 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 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 pre-warmed nuclease-free water.
  • QUBIT ssDNA to quantify yield.

Agarose gel size selection

  • Testing agarose gel purification
  • Prepared >4% agarose gel (Apex multipurpose agarose). 1 g in 25 mL . Microwaved 20s, stop, shake gently, microwave 15s, stop, put on stirrer for 10 minutes.
  • Added 2uL of Sybr Gold to 25mL of agarose solution when the agarose solution have cooled enough to hold for 15 seconds.
  • Poured a gel of about 7 mm thickness with 3-5 mm wells.
  • Loaded different amounts of digested probes (in different wells): 380 ng, 190 ng, 95 ng, 216 ng, 108 ng, 54 ng. (total ~ 1 ug of DNA loaded).
  • Loaded 2.5 uL of low mass ladder (~300 ng).
  • Ran at 50V for 2 hours (120 minutes), the probes were in the bottom quarter of the gel by the end of electrophesis run.
  • Gel image:
 File:Padlock probes agarose gel 50V 120min.jpg
  • The resolution was not very good, but I tried size selection on this gel.
  • I cut out the bottom band & proceeded with the Zymoclean Gel DNA Recovery kit. Eluted the DNA in 20 uL of elution buffer.
  • Yielded ~250 ng, or 25%. Will run TBU PAGE gel tomorrow to check the band size since I could not see the 100 bp on the ladder.
  • Only problem is that the Nanodrop shows high salt concentration...
  • Running out of Agarose, maybe order some of this http://www.affymetrix.com/catalog/129972/USB/Agarose-Hi-Res-Separation-less-than-or-equal-to-1000-bp#1_3 and try to see if resolution can be improved?