first time doing quantitative real time PCR - (Feb/20/2007 )
Hi everyone. I'm working in a real nice place and I'm new over there too. Now I have a challenge "for me", in a sense that I must to develop a quantitative real time PCR. I know the basic but I will love to know more information about how to do an experiment to quantify DNA and how understand the graphics. Thank you
So, i'm new on qPCR also.. but i working on that since 2 months.. and by now it's is going ok. But, qPCR is very tricky...
Here are some advices:
1. Method to quantify RNA/check quality. I use Experion and Ribogreen
2. Design your own primers.. tehre are some software available.. i use beacon
3. Decide the method that you will use, i mean, syber green, taq, beacon.... syber green is the easiest, cheaper...
4. Start to set up the 1PCR experiment... in the begning you need to optimize your qPCR, fold dilution of cDNA, T annealing of the primers....
There is lot of work, but when you look at the results... the reward comes!!!
Take care of the pippeting.. make at least duplicates!