AvrII NEB#R0174

Protocol tips

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	We pooled equal masses of the three aliquots and then enzymatically cut the DNA with six different 6-cutter restriction enzymes, separated the digested DNA on an agarose gel, and cut out and extracted DNA from the sharpest visualized band, an ~2-kb fragment from the AvrII (NEB) digest. We cloned the gel-extracted DNA, amplified colonies, and sequenced them using an ABI 3130xl.

Protocol tips
We pooled equal masses of the three aliquots and then enzymatically cut the DNA with six different 6-cutter restriction enzymes, separated the digested DNA on an agarose gel, and cut out and extracted DNA from the sharpest visualized band, an ~2-kb fragment from the AvrII (NEB) digest. We cloned the gel-extracted DNA, amplified colonies, and sequenced them using an ABI 3130xl.

Publication protocol

We isolated a circular viral genome from buccal and cloacal swabs sampled from an Alaskan black-capped chickadee (Poecile atricapillus) with avian keratin disorder (1) in Anchorage, AK, on 21 December 2010 and preserved it in RNAlater (Life Technologies). We extracted DNA and RNA using a ZR viral RNA kit (Zymo Research). We split the extracted nucleic acids into three separate 3-µl aliquots, leaving one aliquot unmodified. We treated the second aliquot with T7 exonuclease and ExoI (New England BioLabs [NEB]) for 1 h at 37°C, and then 20 min at 65°C to eliminate noncircular DNA. We trapped circular DNA present in the third aliquot by adding 50 µl of 0.8% low-melting-point agarose gel melted in Tris-acetate-EDTA buffer, cooling and solidifying the gel, running under a 100-V current for 1 h, and extracting DNA from the gel (2). Next, we performed a multiple displacement amplification (MDA) using Phi29 polymerase (NEB) on each aliquot and precipitated DNA in isopropanol. We pooled equal masses of the three aliquots and then enzymatically cut the DNA with six different 6-cutter restriction enzymes, separated the digested DNA on an agarose gel, and cut out and extracted DNA from the sharpest visualized band, an ~2-kb fragment from the AvrII (NEB) digest. We cloned the gel-extracted DNA, amplified colonies, and sequenced them using an ABI 3130xl.

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