RT-PCR- help - Polyacrylmide Gel electrophrosis for PCR products (Nov/23/2005 )
Hi,
I am new to this forum. I am stuck with RT-PCR. My aim is to identify isoforms. so i use different primer sets to amplify specific isoforms. When i resolve the rT-PCR products on Agrose gels i get only the bands of my interest. If i resolve the same product on Polyacrylmide gel (PAA) i get extra bands on the higher size. they form a specific pattern on this gel with each of the primer set. i tried changing the annealing tempertaure and also other conditions including the pcr machine. I also sequenced the right sized bands from agarose gel which confirmed the product. Did any of you have faced such a problem with PAA gels? Is there some method to resolve this problem. I did not still sequence the higher bands that i see on the PAA gel. I used PAA gel as the products which i get are on 3-50 nt difference and hence i get better resolution. hope to get some input into my problem thanks in advance vasu
Have you check dissociation curve?
How would that help me ? u mean i have to check the dissociation curve of each product or the primer annealing?
often when using polyacrylamide gels I have observed a ghost band at around double the size of the product
This usually goes away if you heat the product to 95C for a few minutes prior to loading, and load less product on the gel