Real-time PCR machines for DNA quantitation - (Oct/14/2005 )
I was wondering if there is a way to make DNA quantitation measurements using picogreen on the real time PCR machines. It seems that since the machine is making flourescence measurements that perhaps it could make a single reading of flourescence perhaps by changing the cycling parameters of the machine to read just one cycle. Has anyone done this have a idea how feasible this would be with the machine and/or software.
I do not know how to do this, but a tech in another lab here does that all time in the machine where I do my qPCR
I do not know where to go for a protocol, but there should be something in the machine's manual?
I should add that I do not have access to a fluorimeter, which I why I am thinking about this approach.
Like all good ideas, it looks like I am not the first to think about this, here is a reference for a method that I found today. I still would appreciate any input from people who may have some knowledge of this technique. Thanks as always!
http://www.iss.it/publ/anna/2005/1/411119.pdf
http://www.iss.it/publ/anna/2005/1/411119.pdf
No, you are certainly not the first one thinking of this idea. Actually, Stratagene MX3005P has a function of doing single reading acting as a microplate reader.
Yes, I have done that before, using the picogreen kit on a real-time PCR machine. As you set, I just set it to run for one cycle. All you have to do is to make sure that the excitation and detection wave-length (the channels) are set correctly so that it read the fluorescence emitted from picogreen. It worked perfrectly for me.