Problems with SYBR Green assay - bad melting curves and bad amp efficiency (Feb/22/2011 )

adrian kohsf on Fri Feb 25 20:37:18 2011 said:

Hmmm that's a slight complication
I could give you a recipe to make a home-made thermocycler with pans and a stove but real-time instrument is beyond my abilities

BTW, I'm onto something. Looks like the taq from the ABI SYBR green kit isn't a hotstart, sooo maybe all these crazy peaks I see in the dissociation curves are unspecific products?? AB calls the enzymne Fast Taq and includes an activation step but since it is only 2 seconds... ..I'd say this is a regular enzyme.
Will try the Sigma kit that does use a hotstart Taq.

Maddie on Mon Feb 28 15:28:56 2011 said:

I like to have that recipe.
Hmn... what about 3 water bath with different temperature on?

I don't think the hotstart will have such a great effect on it, but I have to say I really not sure... perhaps she can write to the author to ask about that and maybe let us know what happen..