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Dinh:Probes Prep
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== 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 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.
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