Hi everybody, I'm planning to sequence several phytoplasma strains by doing a nested PCR and cloning the resulting amplicons. Standard phytoplasma universal primers pairs will be used (P1/P7 followed by R16F2n/R16R2). I've done this procedure before and it worked stupendously for some samples, got really faint bands for other samples and for a few it didn't work at all.
I know all the samples have phytoplasma DNA because I examined them before with a Real-Time PCR protocol (from this analysis I could tell that phytoplasma DNA concentration varies from sample to sample, and samples with lower Cts where the ones that I could obtain an amplicon without a problem, samples with intermediate Cts resulted in faint bands and those with the highest Cts didn't produce a band).
I want to try again with those that produced faint bands or no bands. What can I do to improve my amplicon yield? I was thinking that after the second amplification I could dilute the product again and run a third one with the same primers R16F2n/R16R2. Has anyone done this? What other things I could do or should I just accept that phytoplasma titer isn't enough for some of my samples? Thank you in advance.
If you are getting only a single band of your target sequence then it is possible to use the product of one reaction as the template for the next. If there are multiple non specific bands then the amplification of your target sequence may not be that efficient and you might see enrichment of your non specific seqences.
In cases where you have low yields, you can either increase the number of cycles or set up a larger volume of reaction and concentrate the product using the PCR cleanup kit.
once I have tried for 5' RACE to run one more cycle to concentrate my cloned product, but unfortunately that was not successful. But you should try once for faint band producing samples .
unfortunately for no bands you cannot do this until you get some template DNA ,
you should go for Direct purification of PCR product(without running AGE) by different available kits that directly purifies PCR Products , and then run small volume if it gives band or not .
Direct purification can be done with faint bands or for faint bands you can run AGE with slightly high concentration of agarose , and low running voltage for longer duration so that you can get a nice band and at this time before casting gel try to increase volume of well so that you can load more running volume .
hope this will solve your issue
All the best
Yes, you can dilute a PCR product 1:100 or 1:1000 and perform another round of PCR on it, even with the same primers. Because of the dilution, there is no need for a purification step between the two PCRs (if you do not have any visible non-specific amplification).
An alternative could be running several identical PCRs in parallel, then pooling the reactions into one purification column. You'll be able to concentrate your sample 5-10 times like this without wasting time on additionnal PCRs. You can always reduce the elution volume from 50ul to 20-30ul which will further concentrate your product. This only works with PCR purification though, not gel purification, as dissolved agarose will clog the column filter if you put too much of it.
Thank you so much! I'll keep in mind all your suggestions, see which kits we have available in the lab and ultimately see which one works best. Martin Chenal I guess I'll try performing another round with both 1:100 and 1:1000 diluted products.
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