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Dinh:Probes Prep May2015
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===DpnII digestion (Day 2)=== * Reagents used: 62 uL DpnII (50 U/uL) * Set up time 30 minutes + 2 hours incubation + 30 minutes cleanup = 3 hr. * To each USER 350 uL reaction, add 40 uL Guide-oligo (NN version), and add 5 uL DpnII buffer = 395 uL. Split into 4x0.2mL PCR tubes (~100 uL each). 94C for 2 minutes -> 0.1C/s to 60C -> 60C for 20 minutes -> 37C for 3 minutes add DpnII To each PCR tube, add a total of 5 uL of DpnII to each set (50U/uL). Use P200 to mix each reaction by re-pipetting 10 times on the thermocycler. * Incubate at 37C for 1 hour. * Aliquot 2 uL of each (A-E) for PAGE-verification * After 1 hour, add mixture of 8 uL water, 1 uL DpnII buffer, 1 uL of DpnII ** NOTE: I forgot to ramp up to 94C and then back to 37C. This would have helped the digestion efficiency more than simply adding more DpnII. Reagent Volume Nuclease free water 8x31 = 248 uL DpnII buffer (10X) 1x31 = 31 uL DpnII 1x31 = 31 uL 10 uL of DpnII mixture to each, use P200 to mix each reaction by repipetting 10 times on the thermocycler. * Incubate at 37C for 1 more hour. * Aliquot 2 uL of each (A-E) for PAGE-verification * Clean up 5x90 uL reactions using 2 ssDNA cleanup columns (Zymo). Use ssDNA column to remove leftover dsDNA! * Yield: {| {{table}} | align="center" style="background:#f0f0f0;"|'''Subset''' | align="center" style="background:#f0f0f0;"|'''Input(ug)''' | align="center" style="background:#f0f0f0;"|'''USER/DpnII, ssDNA column, QUBIT(ng/uL)''' | align="center" style="background:#f0f0f0;"|'''ssDNA elution volume''' | align="center" style="background:#f0f0f0;"|'''Total (ug)''' | align="center" style="background:#f0f0f0;"|'''%yield''' |- | A||14.535||27.4||115||3.151||22% |- | B||11.875||21.6||115||2.484||21% |- | C||15.048||25.2||115||2.898||19% |- | D||15.485||33.6||115||3.864||25% |- | E||12.749||20.6||115||2.369||19% |- | F||13.129||25.2||115||2.898||22% |} * Gel confirmed that DpnII digestion was incomplete, use 100 units and RE_DpnII_V4 (no Ns) to try removing more adaptors. * Gel after USER (O/N), DpnII (1hr), DpnII (1hr) [[File:460k_A-E_prep_USER_DpnII_DpnII_ABC.jpg | 300px]] [[File:460k_A-E_prep_USER_DpnII_DpnII_DEE.jpg | 300px]] {| {{table}} | align="center" style="background:#f0f0f0;"|'''Subset''' | align="center" style="background:#f0f0f0;"|'''Total (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA elution volume''' | align="center" style="background:#f0f0f0;"|'''RE_DpnII_V4 oligo (uL)''' | align="center" style="background:#f0f0f0;"|'''DpnII (uL)''' | align="center" style="background:#f0f0f0;"|'''DpnII buffer(10x)''' | align="center" style="background:#f0f0f0;"|'''Water(uL)''' |- | A||3.1784||116||15||2||15||4 |- | B||2.5272||117||15||2||15||3 |- | C||2.9484||117||15||2||15||3 |- | D||3.9312||117||15||2||15||3 |- | E||2.4102||117||15||2||15||3 |- | E||2.9484||117||15||2||15||3 |} * Gel after final digestion: [[File:460k_A-E_prep_USER_DpnII_DpnII_DpnII_ABCDEE.jpg | 300px]]
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