maximum amplicon size - 550bp too long??? (Feb/23/2009 )

have a question concerning the maximum amplicon size which could be amplified in real time pcr: is approx. 550bp too long?

and what parameters have to be modified during cycling?

thanks in advance

As far as I know, your product should not be longer than 200bp.

Short amplicons yield the most consistent results. Remember that there is no extension so, the amplification will be incomplete if use primers for a 550bp amplicon. Mine are alway between 125-150bp

The amplicons for SYBR assays can be up to 500 bp (didn't try that long though), but you should adapt the elongation time acording to that, should be noted in your mix manual.
Amplicons for probe assays should be as short as possible, because you need short span between primers and the probe.

I am using SYBR green, the ideal amplicon size should be between 50 to 150 bases long. My products is always 50 to 100 bp.

The size of the amplicon shoub be short enough to generate specific amplification.

moljul on Feb 23 2009, 09:09 PM said:

well, 550bp is a little too long when it comes to ideal real-time pcr. But if you are using SYBR Green then 550bp is no problem, as long as you give your reactions enough MgCl (U'd need to increase it, usually), and prolong the extension time. An ext time of 30sec is usually enough. There's basically not much difference between normal PCR and realtime, just that the products are shorter to enable efficiencies nearer to 2 (perfect efficiency) and also to get faster results. But if your standard curves for your gene is pretty good (>1.8/>1.9),then by all means proceed with your real-time. I've amplified up to 800bp, if your primer design and optimization is good, there shouldn't be a problem, no matter what the "ideal" size is.