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Tweety 06-29-2011 06:36 PM

Amplification efficiency
I am doing amplification of different lenght fragments ( I have an unique cDNA wheret I have to amplify two fragments of 474 bp and 2200 bp), do you know if there is a different efficiency in Real time PCR between small and long fragments?

Dr_virologue 07-03-2011 09:52 PM

Re: Amplification efficiency
Of course, the real time PCR doesn't work with a high size, i think the highest size of target gene is 200pb, than i am not sur if your qPCR will give you something.

With Syber green or evagreen (Intercalant florochrome) the first size: 474 bp can work, sur but if you will use the probe (TaqMan), it's sur none of your fragment will work!!

If you need more details, i can help you!

JennYeap 08-08-2011 01:39 AM

Re: Amplification efficiency
Hi, the real-time PCR will be able to detect the long amplicon as well as short amplicon, but its most recommended to amplify short products using real-time PCR. Long amplicons will require more fluorochrome, and insufficient fluorochrome will not give accurate expression levels. hence, its better you re-design your primers to amplify product <300bp. Amplicon size will affect the amplification efficiency indirectly.

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