3 replies to this topic

[The Question]

Hi everybody

I'm new for qPCR and was wondering what can be the optimal target length in qPCR.
One month before I was using primers for target length of 250 and now I'm using the primers for the target length of 60. Can bad target length be problem to determine melting temperature? And can it cause any other problems?

Thanks in advance

[ciyiben]

Hi

In theory, an optimal lenght would be between 100 and 200.

Good luck

[The Question]

ciyiben, on Nov 19 2009, 06:45 PM, said:

Hi

In theory, an optimal lenght would be between 100 and 200.

Good luck

Thanks for reply. Now I've question what can be the problem of shorter target like 60 nucleotides in length, which I'm using.

[Maddie]

Imagine you need to amplify between 200-400bp after your quantification.
If your DNA is degraded, a 60bp qPCR may give you an overestimation of what you have in your tube.
There might be a lot of 60bp fragments but far less 400bp fragments.
When the DNA is pristine, it usually isn't a problem. This is why AB developed Quantifiler with a 62bp amplicon.

To sumarize, I'd try to use a size as close as possible as the the size of the amplicon you will target (unless it's huge of course).

Hope this helps.