Some time ago, we tried to genotype some samples from human saliva, however using the PCR technique ENDPOINT did not work. Then we tried with SYBR Green I, and it worked perfectly. However, now we use EvaGreen ® Dye and nothing works. Could someone guide us?
Hey,
are you using the same MastMix and just the dye is different?
in former times I also used SYBR Green and changed to EvaGreen because it should give better signal.
the MasterMix with this new dye needed to be longer preheated to get active.
Maybe you have the same MasterMix and you just have to do a longer preheating to activate the HotStart Polymerase.
With the new MasterMix (with the new Dye) I have to heat it 15minutes at 96°C.
I hope that helped.
If not you could just take the PCR product and load it on a gel and look if the reaction at all worked and it is just a problem with the dye.
all the best,
Sebastian
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