I'm a bit confused by my cloning PCR results. I have used the TOPO TA Cloning kit and everything seemed to go to plan - I got colonies, I grew them in the indicated liquid culture and then isolated plasmid DNA. I now want to PCR the plasmid DNA with the M13 primers to sequence. However, the PCR product from the M13 PCR should be around 470bp as my PCR product is around 300bp and the M13 primers add another around 170bp from looking at where they bind in the TOPO manual.
Despite this, I am getting a product in the 2500-3000bp region even after doing a gradient PCR and a dilution series of the plasmid DNA template. Does anyone have any ideas what I am missing here? I've checked the M13 primer sequences and they match what is in the TOPO guide so I'm unsure how to progress.
Did you sequenced or did restriction test of DNA before starting PCR ?
No I have not done a restriction digest, [age 12 of the TOPO manual said that I could proceed directly to sequencing?
Do a restriction digest to double-check your insert size and/or sequence your clones. May be you have accidentally cloned another PCR product, which was less abundant. Did you obtain a single amplification product or were there some unspecific background bands visible on your agarose gel?
i must say cloning is not as easy as the protocols say :D i had faced many problems during cloning. the clones that you are getting are false positive ones. but anyhow you can check them for further evaluation, do pcr with gene specific primers as well as with pair of M13 and gene specific primer (this for sure confirms that you got your desired insert in plasmid with correct orientation). perform digestion with restriction enzymes that do not cut your insert. and in the end and most reliable, sequencing; either with gene specific or M13 primer. and probably you can confirm all these on one or two clones to make sure you do not waste your reagents and if you get positive results then go ahead with remaining clones. best of luck :)
Thanks for all your suggestions guys. I will have a go at the gene specific primers/gene specific with M13 primer PCRs to check the insert and perform the restriction digest and let you know how things go.
So far I tried a PCR with my original 16S primers and each of the 4 samples I tested had a band in the expected region and a band at around 4000bp which looks like the whole vector. I tried my forward 16S primer with M13R and all samples had a band around 2500bp, two had two bands between 1000-1500bp and one of these had a product at around 200bp which is what I expected them all to have due to where the primers should bind. I'm have quantified the plasmid DNA and I'm going to try the restriction digest.
I had the same problem. Can i ask if you did a colony PCR to confirm your insert is in the correct direction. If you just choose colonies there is a risk that they contain the wrong, or no, insert. In this case using M13 primers will just amplify the plasmid, sometimes it gives very odd results. Perhaps your 3kb band is the plasmid your using. As suggested above, design gene specific primers and use those.
Hi,
Thanks for your suggestion - I re-ordered a new set of M13 primers and they worked much better - I must have had a dodgy batch. I think the band was just excess plasmid.
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