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-   -   Struggling to amplify DNA with barcoded primers (http://seqanswers.com/forums/showthread.php?t=34121)

firefly2280 09-28-2013 12:12 PM

Struggling to amplify DNA with barcoded primers
 
Dear all

I am using the MiSeq for 16S amplicon sequencing with custom barcodes.

I use the barcoded primers on extracted genomic DNA. However, I have quite low concentrations of starting DNA, sometimes too low for a Qubit to detect. Some samples amplify well with the primers, some dont and some dont at all. It doesnt seem to relate to starting conc as sometimes the ones with undetectable levels amplify well.

I know that there can be random fluctuations in priming efficiency with low conc DNA samples but I dont know how to get those ones that dont amplify to work....

Ive tried touchdown PCR, enrichment PCR and varying annealing temps and template amount added but nothing works. When I do the PCR with the same primers minus the barcodes, they pretty much all work well.

Can anyone help?

Thanks

bnmtthws 09-29-2013 09:47 AM

when we've had similar problems with a barcoded primer set i've simply performed two rounds of PCR - use the primers without barcodes first, and the reamplify with the barcoded primers.

if you want to keep cycle numbers down, you should be able to get away with ~ 10-20 cycles for the first PCR and ~10-20 for the second depending on efficiency.

firefly2280 09-29-2013 11:10 PM

Thanks for your reply. I tried that and got nothing with the first primers after 10 cycles... Might have to give it another go. It works well with a full 30 cycles each but there's no way I want to do that!

bnmtthws 09-30-2013 06:05 AM

i hear you - good luck!

at this point, i don't even bother running out the 1st PCR on a gel - we'll run the results of the 2nd pcr through the qubit and bioanalyzer to check sizing and concentration, and have had pretty good luck so far.

another option is to remove half of the reaction after, say, 10 or 15 cycles and let the rest go to completion to run on a gel - this will give you confirmation that the reaction is 'working' but allow you to use the portion of the reaction that you removed as the template for the next round.

firefly2280 09-30-2013 11:48 AM

Thanks again, I'll have a play with it again. I dont see why I shouldnt be able to get it to work! At least I know Im not crazy and other people can make it work! Thanks


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