For a product that small, I wouldn't worry about trying to change the extension time (30 seconds or less should be just fine).  You should check the annealing temperature for your primers.  If they have a high GC content, then you will need to use a higher annealing temperature.  I've had good luck just using the recommended temp from the primer manufacturer.  Or you can use an online tool like http://tmcalculator.neb.com/#!/

If that doesn't work, then set up a gradient PCR with different annealing temps.

I've seen betaine do magic.  It's marketed as secret "G/C enhancer" compound by various companies.  Try a few different concentrations of it.  I've had cases where it made all the difference between nothing and good amplification. 

Finally make sure your cloning sites are away from the high G/C regions, if at all possible.  It does not just afflict PCR, but also ligation / assembly. 

hi

I am agree with Katie .

you can used 1 ul DMSO in PCR Master mix solution for opening GC loops in your template .

good luck

Hello Reza

The I ul of DMSO will be added to how much master mix? I am using high

GC content primers and I want to improve the reaction (one step rt PCR)?

Hi Aisha

If you worked with QIAGEN OneStep RT-PCR Kit , Q-solution for High GC content .

for example you need 10 ul DDW for total master mix = 25 ul , you should add 9 ul DDW + 1 ul DMSO

good luck

iagree with John that betaine at 1M final concentration is excellent for high GC templates. A mixture of 7 deaza analogue and natural C base will also spoil some of the secondary structural problems associated with high CG and allow better amplification, Ref above I think that Q solution is betaine or a mixture of betaine and DMSO but Qiagen will not confirm its constitution ( very reasonably)

Thank you all very much for your helpful answers! I will try adding betaine and DMSO in the mix and also improving the Tm. Later I´ll write how it goes.

Thanks!!

High-GC means more energy required for breaking H-bonds. Accordingly, I would elongate the denaturation time to provide more heat, and maybe higher temperature like 96-98C could also be useful. Opposite to denaturation, extension is a process for new bonds formatting, so heat will release. As a consequence, a lower temperature with shortening time may help.