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Cloning with pBR322 and two restriction enzymes

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Cloning with pBR322 and two restriction enzymes

Postby SamVG » Wed Apr 13, 2011 4:34 am

I was wondering what happens when you cut a plasmid (pBR322) with two restriction enzymes and then insert a piece of DNA to be cloned. Is the piece from the plasmid lost?
e.g. cut a plasmid with EcoRI and BamHI which are 0.4kb's apart and then insert a piece of DNA for cloning which is 1.5kb's long will the resultant be 1.9kb or 1.5kb's?
I have searched all my textbooks but I can only find examples where the pasmid is cut with only one restriction enzyme.
Thanks so much,
Sam :)
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Postby protold » Wed Apr 13, 2011 5:42 am

Hola, the most usual method is separate both bands from digestion of plasmid in an agarose gel, cut and extract from agarose the digested plasmid band, and ligate with the new insert. If you want to do it in the same tube of digestion, after it you have to inactivate restriction enzymes, and add the new fragment and ligase, but the problem will be a high bacground of the religated pBR322 with his portion of MCS that is in the tube . Buena suerte
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Postby JackBean » Wed Apr 13, 2011 12:14 pm

First of all, it doesn't matter that much, how many enzymes you use, but rather on how many restriction places you get. E.g. you can add 10 REs, but nine of them won't cut either once and only one will cut once, while on other hand, yu could use only one enzyme, which cuts 10 times. And since the vector is circular, the number of fragments equals to number of restriction sites, while for linear DNA it would be n+1.

The size of whole plasmid is 4.36 kbp and the difference between these two restriction places is about 370 bp, so you will get approx. 4 kbp and 370 bp fragments.
As protold said, usually after restriction, you separate the fragments and thus get only the (in your case) 4 kbp and this use for ligation with your insert. However, you don't need to separate them, but instead you could dephosphorylate it and use for ligation, thus it won't ligate back with the cut-off-part and either it won't self-ligate ;)

Cis or trans? That's what matters.
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Postby Papaver » Tue Aug 16, 2011 1:05 pm

It depends on the aim of your experiment / cloning.
If you want to clone a specific fragment into plasmid you need to find enzymes that cut once. Imagine you take an enzyme that cuts near the cloning site but also in the middle of an orf (e.g. antibiotic resistance gene). Then you would lose a big but important part of the plasmid. That means to have more site the enzymes cut you risk to lose necessary DNA regions.
You always look for an unique site and either choose to clone with one or with two different restiction enzymes. The latter is usually better for determine the direction of your fragment.
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Postby jophly » Fri Aug 19, 2011 3:35 am

the problem will be a high bacground of the religated pBR322 with his portion of MCS that is in the tube . ...
Last edited by honeev on Fri Aug 19, 2011 6:04 am, edited 1 time in total.
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