plasmid insertions

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plasmid insertions

Post by supaboy » Mon Mar 15, 2010 1:32 am

Hey, sorry im just a lil stuck on a few things relating to plasmids etc.
For cloning a nucleotide insert into a plasmid, what are the advantages that a multiple cloning site have over an individual restriction site? Is it cause this allows flexibility when inserting gene fragments into the plasmid vector and that restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA?

Secondly if you have a recombinant expression plasmid termed pGEX-T-SARM which was constructed by ligation of an insert containing the SARM gene into the pGEX-T (bacterial expression plasmid) multiple cloning site, if you transform pGEX-T-SARM into bacteria, what is the mechanism whereby addition of IPTG will induce the expression of GST-SARM?

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Inland Taipan
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Post by JackBean » Mon Mar 15, 2010 3:15 pm

all the restriction recognition sites occur with some probability all around the DNA, so if you had only one restriction place in your vector (meaning in the place where you want, of course there are plenty of other restriction sites all around the plasmid), there is chance, that your insert would be cut with the same restriction endonuclease. Also, if you use two restriction enzymes, you can insert it in correct orientation through complementary binding of sticky ends.

I forgot the second answer. Look for lac operon

Cis or trans? That's what matters.

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