Protocol Online logo
Top : New Forum Archives (2009-): : PCR, RT-PCR and Real-Time PCR

PCR using CDNA as template - (Aug/30/2011 )

I have been trying to amplify a 3'UTR region of an RNA virus. SO i initially made the cDNA and now doing end-point PCR to amplify the UTR region.My primers are to the ends of the UTR region along wiht the restriction sites attached to the prrimers.Total product size 616bp.
I have been getting a clear band in the b-actin control run in the same run, but not the band of my interest. but there are loads of primer dimers at the end!!any suggestions???is it just the annealing temperature?


What annealing temperature are you using? Have you analysed your primer sequences for dimers and/or hairpin structures? and what is your melting temp of your primers? If not, a great program is oligo analyzer. just punch in your primer sequence and it gives you all the information you need