AvaII NEB#R0153

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	Digestion reactions were performed at 37°C for 30 min. Reaction buffer was based on the NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate) in which commercially available restriction enzyme AvaII (NEB) has 100% activity. AvaII was used in the amounts of 100, 10, 1, 0.1 or 0.01 pmol of dimer in 1 μl volume. Results were visualized by native polyacrylamide or agarose gel electrophoresis.

Protocol tips

Digestion reactions were performed at 37°C for 30 min. Reaction buffer was based on the NEBuffer 4 (20 mM Tris-acetate, pH 7.9, 50 mM potassium acetate, 10 mM magnesium acetate) in which commercially available restriction enzyme AvaII (NEB) has 100% activity. AvaII was used in the amounts of 100, 10, 1, 0.1 or 0.01 pmol of dimer in 1 μl volume. Results were visualized by native polyacrylamide or agarose gel electrophoresis.

Publication protocol

"The RNA strand of RNA/DNA heteroduplexes was invitro transcribed from annealed DNA duplexes using T7 RNA polymerase prepared locally. The RNA was gel purified, dephosphorylated using FastAP (Thermo Scientific™), silica column purified (AAbiotechnology), 32P radiolabeled (T4 PNK Thermo Scientific™) and subsequently again silica column purified. The DNA strand of the heteroduplexes was ordered as a synthetic oligonucleotide, gel purified and radiolabeled (T4 PNK Thermo Scientific™). Next, hybrid DNA/RNA and dsDNA duplexes were prepared by mixing complementary strands with slight excess of the non-radioactively labeled strand (1.05:1.00) and annealed in the thermocycler (from 90 to 20°C within 3 h). Digestion reactions were set up in 12 μl volume using 96 fmoles of duplex per reaction, with SUPERase•In™ RNase Inhibitor 1 U/μl, in the buffer recommended by the manufacturer. Enzymes were used at a maximum concentration compatible with glycerol content lower than 4.75%. MvaI, HindIII, PvuII (Fastdigest™ series) and BcnI enzymes were from Thermo Scientific™. AvaII, HinP1I and EcoRV were from New England Biolabs.

Reactions were incubated in the thermocycler for 16 h in 37°C than stopped by addition of 14 μl of loading dye (95% formamide, 25 mM ethylenediaminetetraacetic acid (EDTA)). Standard denaturing polyacrylamide gel electrophoresis in 8 M urea was carried out. Before loading on the gel, samples were incubated at 80°C for 10 min. Cleavage products in RNA/DNA digestion reactions had to stem from the RNA/DNA heteroduplexes and not from contaminating dsDNA template, because the template was 20 bp longer than the heteroduplex. Moreover, only one radiolabeled fragment was observed, whereas two would be expected if the cleavage product was derived from transcription template dsDNA."

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