GAPDH primer design and efficiency problems (new to rt-pcr)
Posted 06 July 2010 - 07:07 AM
Recently, I designed a set of GAPDH primers using Primer-Blast but for some reason I am getting extremely high efficiencies when I run a standard curve with these primers. I was wondring if someone could look over the primer sequence and let me know if there may be problems with the sequence itself.
The sequence is:
I did a gradient intially with these primers from 55-65 oC and they seemed to be fine at all temperatures but when I ran a 10x standard curve the efficiency was around 135%. I would imagine trying to design reference gene primers would be the easier task but I guess I've been proven wrong.
If anyone could please help me out that would be great.
Posted 06 July 2010 - 08:01 AM
Posted 06 July 2010 - 08:08 AM
I also designed my GAPDH by primer-blast at NCBI.
use 1.5ul of 10pmol of working solution primers for every 50ul reaction.
Are you doing Real-Time PCR?
Posted 06 July 2010 - 08:27 AM
Yes, I am doing real-time PCR and I had been using 500nM final concentration of each primer in those reactions.
When I did normal gradient PCR, I used 2uL of a 10uM primer solution in a final volume of 50uL...do you think this is too high?
Posted 08 July 2010 - 10:40 AM
I did a nucleotide Blast with these sequences and I'm getting some E values that are pretty low under genomic sequences...is this cause for any big concern?