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question about plasmid linearization


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#1 lxyape

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Posted 27 December 2004 - 01:10 AM

I've isolated plasmids from bacteria from environmental sample and want to determine the size of them. But I don't know how to choose nuclease to cut them. Please give me some suggestion.

#2 leahf

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Posted 27 December 2004 - 06:18 AM

as far as i know, its mostly luck, but here are some guidelines:

1.six-cutters (restriction enzymes that recognize a site of 6 specific bases) cut more rarely than four-cutters, so are a better choice to start with - (four cutters are more likely to give you many small fragments).

2. composition of restriction site - if your bacteria is very GC rich, AT-rich recognition sites will be rare, and vice versa.

A few very common 6-cutters are EcorI, BamHI, XbaI, but there are dozens more. Most people just use whatever is avialable in their refrigerator.
Specifications of the restriction site of each enzyme you can find in comercial catalogs, such as Biolabs and Fermentas.

Luck!

#3 lxyape

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Posted 27 December 2004 - 04:56 PM

Thanks, I'll try to cut them by Ecoró˝.




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