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Does EthBr turn restriction enzymes into nickases?

I'm looking for a way to create a nick at a specific point in a closed circular plasmid, to see if increased DNA flexibility facilitates DNA uptake. New England Biolabs now sells specific nickases, but their site preferences aren't very convenient. I also have an old paper (Shortle et al. 1982 PNAS 79:1588) describing use of ethidium bromide to convert restriction enzymes into nickases. So I think I'll just do a simple test. Here's the relevant paragraph from that paper:
Restriction Enzyme Nicking Reactions. Covalently closed circular pBR322 DNA was nicked with restriction endonucleases HindIII, Cla I, or BamHI by incubating 10 pkg of plasmid DNA in a 100-Al solution of 20 mM Tris HCl, pH 7.8, 7 mM MgCl2, 7 mM 2-mercaptoethanol*, gelatin* (100 Ag/ml) and a concentration of ethidium bromide (50 ug/ml, 75 ug/ml, or 100 ug/ml, respectively) determined by titration to give an optimal level of nicking. An amount of restriction endonuclease was added sufficient to convert 50-90% of the input DNA to an open circular form on incubation at room temperature for 2-4 hr. The nicking reaction with the EcoRI enzyme consisted of 100 mM Tris'HCl, pH 7.6, 50 mM NaCL, 5 mM MgCl2, gelatin* (100 ug/ml), ethidium bromide (150 ug/ml). Reactions were stopped by addition of excess EDTA followed by phenol extraction and ethanol precipitation.
I have lots of supercoiled plasmid that has one EcoRI or BamHI or HindIII site, and a big bottle of 1 mg/ml EthBr. So I'll just set up a series of digests with different concentrations of EthBr and restriction enzyme and the standard digestion buffers*, and run a gel to see what I get.

*In the old days we used to add mercaptoethanol and gelatin to stabilize our restriction enzymes, but I won't bother.

1 comment:

  1. If I understand you correctly your looking for an enzyme that will nick your supercoiled plasmid at a specific position and make it a relaxed circular molecule?
    In that case why not use PCR with a HF taq that can amplify a complete plasmid (like turbo Pfu from stratagene) to do so. You could set the nicking point using a primer, sure it'll take much longer then a restriction reaction but i think its better then experimenting with EtBr :)

    Ido.

    P.S
    Don’t have any business interest in stratagem (or any other company for that matter)
    P.P.S
    If you already done it how did it go?

    ReplyDelete

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