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Can we add a restriction site to a gene?

DNA gene transformation restriction site plasmid

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#1 FogtBiotech

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Posted 12 March 2014 - 07:50 AM

I'm a student in my high school biotech class, and we are currently trying to insert a gene into a plasmid. Before we PCR this gene, we would like to add restriction sites to both ends of the sequence to create "sticky ends" that will fit into the plasmid more easily.

Is there any way we can do this? And how would we carry it out?



#2 phage434

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Posted 12 March 2014 - 07:58 AM

You can easily add a restriction site to ends of your fragment, by expanding your PCR primer. You simply have to add the restriction site recognition site 5' of the PCR binding tag. You should also add another 4-6 bp of "junk" DNA 5' of that restriction site to allow the restriction enzyme to cut effectively.

 

You should (of course) choose enzymes whose recognition site is not present on the fragment you are amplifying. You should choose different enzymes for each end. It would be best to choose enzymes leaving a 4 bp overhang, and ones which can be heat-killed.







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