Hi, a bit more information to sufficiently answer your question might be helpful. How did you proceed after PCR? Did you dilute your final PCR products? Or did you perform a dilution series before PCR? And what do you wanna find out? The number of replicates or the concentration,...

Thanks

Bastian

Hi Edisa, Still the information provided by you regarding your experment is not sufficient. Can elaborate what is the aim of your experiment and what kind of analysis your looking for. Still i will suggest you something if your aim is to study diversity among the samples in study.

I think there is no need to dilute the pcr product unless and until the concentration of pcr product is too high. In case of highly concentrated product there is problem to distinguish between very fine bands in dgge gel. Some times not too much concentration but strong enough is need if your looking to identify dominant microflora in the samples.

For the second bit of your question I will suggest you to manually score the bands on gel in each lane and put the score as 1 if band is present in both the lanes or put 1 where the band is present and pur 0 when corresponding band is absent is second lane. Feed this scoring data to ntsys software to generate the similarity matrix to compare between two lanes. Also there are many software available to score the bands on gel automatically. Such as biocompaer and image lab etc.