FastDigest MlsI

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	We digested the RCA products of plasmid DNA pUC19 and pBluescript II SK (+) using respectively FastDigest MlyI (0.5 U/μl, Fermentas) which recognizes GAGTC(5/5)∧ sites, and FastDigest MlsI, which recognizes TGG∧CCA sites. We performed the reactions at 37°C, in 1× Fast Digest buffer, for 2 h to ensure a complete digestion.	We loaded the digested products and their corresponding undigested RCA products, on 1.5% agarose gel (0.5× Tris-Borate-EDTA buffer (TBE) which is made of 44.5 mM Tris-borate, 1 mM Na2EDTA dissolved in deionized water with the addition of etidium bromide (1 ng/μl, Sigma Aldrich)) and we analyzed them by electrophoresis, at 120 V for 2 h. We acquired images by UV trans-illumination (UVITEC) and we analyzed them by the software ImageJ.

Protocol tips
We digested the RCA products of plasmid DNA pUC19 and pBluescript II SK (+) using respectively FastDigest MlyI (0.5 U/μl, Fermentas) which recognizes GAGTC(5/5)∧ sites, and FastDigest MlsI, which recognizes TGG∧CCA sites. We performed the reactions at 37°C, in 1× Fast Digest buffer, for 2 h to ensure a complete digestion.
Downstream tips
We loaded the digested products and their corresponding undigested RCA products, on 1.5% agarose gel (0.5× Tris-Borate-EDTA buffer (TBE) which is made of 44.5 mM Tris-borate, 1 mM Na2EDTA dissolved in deionized water with the addition of etidium bromide (1 ng/μl, Sigma Aldrich)) and we analyzed them by electrophoresis, at 120 V for 2 h. We acquired images by UV trans-illumination (UVITEC) and we analyzed them by the software ImageJ.

Publication protocol

We incubated pUC19 plasmid DNA (1 ng/μl, Invitrogen) with nicking endonuclease Nt.BspQI (0.5 U/μl, New England Biolabs (NEB)) in 1× NEB3 buffer for 2 h at 50°C, and then we heat inactivated the enzyme incubating the reaction mixture at 80°C for 20 min. We then amplified the resulting nicked DNA (0.25 ng/μl) by using phi29 DNA polymerase (0.5 U/μl, Fermentas) for 24 h at 30°C in a 1× phi29 reaction buffer (33 mM Tris acetate, 10 mM magnesium acetate, 66 mM potassium acetate, 0.1% (v/v) Tween 20 and 1 mM Dithiothreitol (DTT); Fermentas) containing dNTP mix (1 mM each; Fermentas) and increasing concentration of single stranded binding protein T4 gene 32 (0–100 ng/μl, NEB). We also performed nicking and amplification reactions with pBluescript II SK (+) (with and without T4 gene 32 protein) in the same conditions. We digested the RCA products of plasmid DNA pUC19 and pBluescript II SK (+) using respectively FastDigest MlyI (0.5 U/μl, Fermentas) which recognizes GAGTC(5/5)∧ sites, and FastDigest MlsI, which recognizes TGG∧CCA sites. We performed the reactions at 37°C, in 1× Fast Digest buffer, for 2 h to ensure a complete digestion. We loaded the digested products and their corresponding undigested RCA products, on 1.5% agarose gel (0.5× Tris-Borate-EDTA buffer (TBE) which is made of 44.5 mM Tris-borate, 1 mM Na2EDTA dissolved in deionized water with the addition of etidium bromide (1 ng/μl, Sigma Aldrich)) and we analyzed them by electrophoresis, at 120 V for 2 h. We acquired images by UV trans-illumination (UVITEC) and we analyzed them by the software ImageJ.

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