cloning - (Dec/13/2007 )

I am trying to clone a fragment some restriction sites into a plasmid that I have partially digested with NotI. Apparently, the fragment prefers one site to the other. Anyone has any good suggestions as to how I can prevent this? I keep getting it cloned into the wrong site.

Any advice is welcome. thank you

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are both sites in a circular plasmid? Any obvious difference between the two sites? NotI requires about 9bp flanking both ends of the restriction site for the enzyme to cut the site. What kind of cell did you use to grow your plasmid in? Could this be some strang methylation problem?

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We have found a similar thing during some weird cloning steps. Enzymes do seem to have preference of one site over other. It is probably dependent on the vector or a particular sequence beside these sites. I got no clue.

You could try it a couple of times, but if its the same thing over and over, I would plan a new strategy.

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I have tried a couple times and it happens all the time. What kind of new strategy will you use?

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I would try to design primers with sites not present in the fragment and try to clone it into the vector. this should be simple digest and not partial digestion.

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There are two things to try.

1) Ethidium bromide intercalates into dsDNA, thus inhibiting or reducing restriction endonuclease activity. (This is mainly a way to get just one cut out of multiple sites, but sometimes it changes the preferential site.)

Set up a 50 µL master mix with 5 µL of 10x buffer, 2 to 5 µg of plasmid DNA and 5 Units of restriction enzyme.
Split the master mix into 5 microfuge tubes and add ethidium bromide to a final concentation of 0, 0.02, 0.01, 0.002 and 0.005 mg/mL. Incubate at 37°C for 20 minutes and analyze products on a gel.

2) In the days before plasmid prep kits that use cartridge purification, I noticed that preferential sites frequently changed after being processed through a cesium chloride/EtBr gradient, followed by phenol/chloroform extraction and ethanol precipitation.

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I am not able to do any simple digest because all restriction sites in my vector are duplicated.

I used the ethidium bromide method, gel extracted the linearized vector, CIP and ligated to my fragment. How can I force the vector to cut at the right site?

Thanks

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does it help if I add ethidium bromide immediately after adding my restriction enzyme? I used to add ethidium bromide before adding my restriction enzyme.

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You might try a partial CpG methyltransferase (NEB) treatment to see if it prefers the same Not I site. Then cut with Not I.

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Mutate out the problematic site with the Quikchange method, or by PCR around the entire plasmid, followed by digestion with some new restriction site you have inserted.

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