concentration of primers - (Nov/15/2006 )
Anyone can help!!
I m doing my Q-PCR currently. The problem is the negative control (without templates) has been amplified at least to some extent (with Ct value about 25-30). Then I attemptted to dilute down my primers to see if this can solve the problem (I made 1/50, 1/100, 1/200, 1/300, the original concentration of primers is 1μg/μl). However, after running on real-time machine, amplified primer controls still appeared I used SYBR GREEN as my detection method) Could anyone be able to help me potentially.
Thanks a lot indeed
Run on a gel or do a melting curve to make sure this is an issue with dimers and not a PCR contamination.
Thanks for your reply tap, But the problem is I added the dissociation curve in the Q-PCR reaction and relatively strong peaks differerent from the amplicon I interested appeared. How could I do
You should first of all run a gel. Do you get a clear, defined band? How big is it? Is it primer dimers or is it an actual PCR product? Put together this information with the melting curve (does the peak have low or high Tm? Is it also well defined, or does it look kind of blurry?), so you can decide whether you have an issue with primer dimers (best thing then redesign primers), or contamination (change all your reagents and cross fingers)...
You may have to consider redesigning your primers. This is not uncommon with sybr green just be happy you did the controls and did not waste time with an artifact and many people seem to do.
Thanks for your advise Tap14. Will do