Q-PCR questions: Primer design and melting curve - (Apr/06/2006 )
As for the primer and template amount, I have been adding as provided in the protocol from Biorad. I did a meting curve and a got single peak. With GAPDH I am not seeing a signal with no template and not RT, that is case only when I use TNFa and IL-1 primers.
I have also asked a couple times, and this is important: what have you done to optimize primer and template amount that you are adding?
if you are getting a signal with no template and no RT controls, then I think primer-dimer needs to be ruled out. Have you read my posts?
did you run a melting curve? if so, what was the result?
and yeah, there could be some other contamination. I would also recommend running a straight PCR with various components to see if you can isolate a source of contamination
well then that's probably primer-dimer
before re-designing your primers, I would work a little to optimize. it can make a really big difference, believe me
Could you explain me what xactly you mean by optimize the reaction
before re-designing your primers, I would work a little to optimize. it can make a really big difference, believe me
I mean, don't just add the default concentrations/amounts of primers and template
try a gradient of each
you will have to do it anyways, when you want to obtain efficiency data for your results. but, here is an example for you. for one of my primer pairs (I look at about 8 total) I have found that adding double completely gets rid of my product and I only get dimers; if I add primer in a fairly narrow concentration range, my product is the only thing I get and it's very reliable
melting curves can tell you this, but a gel is also good...for example, I would add primer in various concentrations (say 50,100,200...up to maybe 600?), run the reaction, and run a gel...you will probably find a range that works well for you.
if not, back to the drawing board and new primers
have you used published primer sequences? or have you designed them from scratch? did you use any software to check them, that can show likely secondary structure?