I am considering having students amplify whole plasmids through PCR. I've never tried 'whole plasmid pcr' and so have no experience with it. I'm wondering if it is efficient or practical.
We do "around-the-world" PCR of plasmids, for example when doing site-directed mutagenesis.. we have done this for plasmids ranging from 6-10kb. Pfu polymerase works well for this, using 2 primers that are reverse complements of one another. In those primers, we can introduce mutations at a specific site, but the PCR will work for your purposes just as well with primers 100% complementary to the plasmid. The primers are usually quite long, around 35 nt.
Amplifying an entire ~5000 bp plasmid may be difficult depending on the kind of DNA-dependent DNA polymerase you use. You would most likely first want to make sure the plasmid is linearized (by single-site cutting restriction enzyme digestion), also you would probably want to use a plasmid with a known insert to get efficient amplification of just that region (1200 bp or less). But, then again, you may want to try exactly what you said using a forward and reverse primer that are complementary to disparate regions of the plasmid - but be sure to linearize the plasmid beforehand so as to not stall the onset of the PCR on account of it being impeded by waiting for 100% thermal breakage of each double-stranded circle. In general, plasmids seem to 'love' PCR...
So, in the end, I would say it's worth a try - just linearize first, then proceed.
You could compare circular (supercoiled) plasmid vs. linearized plasmid PCR amplification to add another point of interest here.
Then - of course run the products out on a 1% agarose gel afterwards (stained with e.g. GelRed) and visualize the size of the product in each case as compared alongside the original supercoiled and/or linearized plasmid ...
We do "around-the-world" PCR of plasmids, for example when doing site-directed mutagenesis.. we have done this for plasmids ranging from 6-10kb. Pfu polymerase works well for this, using 2 primers that are reverse complements of one another. In those primers, we can introduce mutations at a specific site, but the PCR will work for your purposes just as well with primers 100% complementary to the plasmid. The primers are usually quite long, around 35 nt.
Hi, from my little experience as medical microbiologist, plasmid is very effective for PCR amplification, because some bacteria like E.coli habour large plasmid-mediated antibiotic resistance (e.g AmpC beta lactamases). But is good to quantify the isolated plasmid, before the PCR reaction.
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