I have tried adding 1X SYBR Gold to a PCR mix (Hot Start Qiagen) with primers and DNA that I know work with a normal PCR.
However, I did not get any amplification curve (detection at 550nm), nor did I get any bands after running the PCR reactions on a gel.
Any ideas what might have gone wrong? What might have inhibited the PCR reaction, and how to rectify it?
Thanks
So, I found out that SYBR GOLD at a concentration of 1X does indeed inhibit PCR reactions. On diluting SYBR Gold 1:100 times, I was able to use it in the qPCR, after adjusting the amount of MgCl2.
Thanks everyone for the answers!
You can confirm that SYBR GOLD is inhibiting PCR using several dilutions of the reagent. It is rare, but may happen with specific DNA sequences and high concentrations of SYBR GOLD
You have to use SYBR Green 1. SYBR Gold bind too tightly DNA and is not suitable for QPCR.
- Badges
- Science topic