HI

In most cases the fluorescence obtained during qPCR with Intercalating dyes is result of sum of aplicon fluorescence plus unspecific amplifications ( i.e primer dimer ). Thus if you want to use these data for quantification you'll have a very important bias, specially in samples with low target levels.

After the PCR you have to review the Tm in melting program analysis, if you detect more than one peak the high Tm is from your PCR product, the other ones will have low Tm.

If you want to obtain only fluorescence from your target amplification, after the elongation step you have to include one additional step with a temperature high than the primer dimer product but low than the Tm of your product.

One example for Lightcycler ( capillary ) :PCR, 95ºC 15 s / 60ºC 20s / 72ºC 20 s / 87ºC 20 s ( Fl reading ) I use this procedure for Pseudomonas,

best

Designing primers that generate a very long amplicon may lead to poor amplification efficiency. Ideally, amplicon length should be 50 to 150 bases for optimal PCR efficiency. In cases in which longer amplicons are necessary, optimization of the thermal cycling protocol and reaction components may be necessary.

Give it a try. Test efficiency to be sure about the performance of the reaction. Best!

This is a rather long product. Usually I use somethin between 80 and 120, if no shorter products are possible, I go up to 150 in maximum. The longer your amplicon gets, the more negative side effects (lower amplification efficiency, suboptimal reaction conditions such as temperature, amplification time, salt and primer concentrations) will bias your result. Longer amplicons are possible but they need a careful adjustion of the reaction conditions.

Agree - I would recommend using primers that amplify a 65-120 bp amplicon. You may be able to amplify a 320 bp fragment using SYBR from cell line-derived cDNA, but there is increased likelihood of non-specific amplification. If you progress your work to FFPE/tissue-derived RNA, the extensive fragmentation in the RNA will mean you will get primer dimerisation as the 320 bp fragment will likely to be either fragmented or chemically modified. I would suggest resigning your primers to a smaller amplicon - ensure the amplicon spans an exon boundary, if possible, to prevent amplification of genomic DNA.

I would agree with Christian. I have generated 300 bp products with real-time sybr green PCR without problems. You obviously have to extend extension times and there is, however, more potential for artefacts The main reason I have for not doing this routinely is that beyond 180-200 bp, the products all end up having very similar melting curve profiles, and it can become difficult to distinguish between products.

Ian

Dear colleagues

Thank you all for answers

thanks, but I have to use these primers and I can't design primer elsewhere. I have to try this one. thanks again.

Hi

i'm agree with Ian Teo, I usually do it for microbial targets without any problems because vPCR often needs long amplicons. However I experimented some problems with certain master mixes and quite long amplicon ( > 500 bp).

In order to detect only your product during qPCR , you can add one additional reading point at your PCR program, 5 s at Tm-3ºC ( probably each thermacycelr needs diiferent times or fl reading temperature )

best

I amplified an amplicon of 310bp using SYBR Green chemistry. If you make necessary changes regarding the duration of temperature steps, you will come up with excellent results. I would suggest you to test the amplified product on agarose gel for any kind of weak or nonspecific amplification apart from running melt curve. All the best!!!

Hi Francesc Codony

Thanks a lot for your help. Can you please clarify your answer? you said: I can add one additional reading point at my qPCR program, 5 s at Tm-3ºC ,I don't know the reason! and how do i add ? (to PCR cycles or the end of PCR) which step?

Can you please explain it? thanks in advance.

HI

In most cases the fluorescence obtained during qPCR with Intercalating dyes is result of sum of aplicon fluorescence plus unspecific amplifications ( i.e primer dimer ). Thus if you want to use these data for quantification you'll have a very important bias, specially in samples with low target levels.

After the PCR you have to review the Tm in melting program analysis, if you detect more than one peak the high Tm is from your PCR product, the other ones will have low Tm.

If you want to obtain only fluorescence from your target amplification, after the elongation step you have to include one additional step with a temperature high than the primer dimer product but low than the Tm of your product.

One example for Lightcycler ( capillary ) :PCR, 95ºC 15 s / 60ºC 20s / 72ºC 20 s / 87ºC 20 s ( Fl reading ) I use this procedure for Pseudomonas,

best

Dear Francesc Codony

Thank you very much. I'll try your suggestion!

Best regards