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Restrction digest on a plasmid--how many fragments?

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Restrction digest on a plasmid--how many fragments?

Postby biology_06er » Sat Jun 16, 2012 12:41 am

Hi there,

Soo, cloning for about a whole year I kinda just thought about this question!! ....If I have a vector (pBS) for example...and my gene of interest that I wish to clone into it and then GOI has a EcoRI site at N-terminal and BamHI at C-terminal...so I would cut my GOI with those enzymes, and like wise I would cut pBS with those enzymes (assuming it had those sites)...no my question is, why I cut pBS with the two enzymes, and run a gel..should I not end up with two bands on my gel..the linear pbs which i will clone into but also the piece that I cut out as I cut twice?...but in all the times I have done this, I only ever see one band..and I was told..you won't be able to tell if it''s cut twice..only once (when compared to a uncut plasmid). is it that the spacxking is so close..the dna fragment that is lost..is too small to see on the gel..

I've have searched for this online..but exmaples only talk about cutting the plasmid with one enzyme

cheers,
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Postby canalon » Sat Jun 16, 2012 12:57 am

You are right. In a multiple cloning site, the restriction site are usually within less than20 to 30 bp of one another. Look at your plasmid map. And remember that short band are not only fast, they are also very faint (you have one molecule of EtBr every 10 or 11 nt, so for n plasmid you have at best 2n molecule of EtBr linked to your short fragment and someting like 500n or 1000n in the plasmid. When you adjust the sensitivity of your camera, the fain signal will disappear)
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