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Sequencing RT-PCR product


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#1 Ubiquitous

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Posted 14 March 2013 - 07:59 PM

So I have a validated set of primers for rtPCR from Biorad that contains SYBR green. If I do rtPCR, can I use the rtPCR product after purifying it with a Qiagen PCR purification kit? Also, I'm under the impression that the sequencing company needs primers to do sequencing, do I just use the PCR primer or one of the "universal" primers the company has?



#2 bob1

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Posted 14 March 2013 - 08:11 PM

By rtPCR you mean real time- or reverse transcription- PCR? If the former, to avoid confusion it is better to use quantitative PCR (qPCR).

As far as I know primers do not contain sybr green, the sybr agent is much like ethidium bromide in that it relies on intercalation between the DNA strands at a particular density per bp, and as such sybr is added as part of the PCR mastermix. It is possible however to get primers that are fluorescently (fl) tagged such as Taqman assay primers.

In practice it shouldn't matter whether the primers are tagged for use in sequencing, though I have never tried it. I can imagine that a Fl tagged primer may obscure the signal from some of the initial base calls if used though.

For sequencing, yes the company does require primers, you need to set up a tube that contains the target DNA and ONE primer only - if you have both primers in the same tube you will get mixed signal as the forward and reverse regions get sequenced simultaneously (think about how DNA is replicated and the orientation of the two strands).

The universal primers are only for sequencing off plasmids (and even then, only for some plasmids) if your product is not contained in a plasmid you need to supply the correct primer.

#3 Ubiquitous

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Posted 14 March 2013 - 08:27 PM

By rtPCR you mean real time- or reverse transcription- PCR? If the former, to avoid confusion it is better to use quantitative PCR (qPCR).

As far as I know primers do not contain sybr green, the sybr agent is much like ethidium bromide in that it relies on intercalation between the DNA strands at a particular density per bp, and as such sybr is added as part of the PCR mastermix. It is possible however to get primers that are fluorescently (fl) tagged such as Taqman assay primers.

In practice it shouldn't matter whether the primers are tagged for use in sequencing, though I have never tried it. I can imagine that a Fl tagged primer may obscure the signal from some of the initial base calls if used though.

For sequencing, yes the company does require primers, you need to set up a tube that contains the target DNA and ONE primer only - if you have both primers in the same tube you will get mixed signal as the forward and reverse regions get sequenced simultaneously (think about how DNA is replicated and the orientation of the two strands).

The universal primers are only for sequencing off plasmids (and even then, only for some plasmids) if your product is not contained in a plasmid you need to supply the correct primer.



Thanks for clearing this up for me. Yes I meant qPCR. And no the primers don't contain SYBR green, which is part of the master mix, you're right. Made some errors describing what I want to do because this is the first time I've ever tried PCR.

#4 bigudukaz

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Posted 27 March 2013 - 01:42 AM

You should ask the company whether the SYBR die interferes with their sequencers. The safest way to do so would be purifying the product threw the column (i have used ones from Thermo Fisher - GenJet PCR purification kit) - id does what it says :)




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