Titration of Mg2+ in RT-PCR - (Dec/22/2005 )
I'm new to RT-PCR and I am not getting and bands. I've designed primers spanning and intron, performed RT using SuperScript II following the protocol supplied. One of the things I wanted to try is tirtation of the Mg2+. If I change this concentration do I need to change anything else in my reaction mix?
Also, I need to include a positive control which would be best and do I need to design my own primers for the control? I'm working with plants.
um, yeah, you really need positive controls. your problem may be with degradation and this could help you pinpoint the source, if you are getting no bands at all. RNA is notorious for degradation if you don't jump through all the little hoops to prevent it (I am sorry if you already know all this, but from your post I would look at this aspect first)
are you familiar with Ambion's website? they have many excellent technical references and suggestions for RT-PCR, and working with RNA-related applications in general. sometimes you have to wade through the web page a little, but they are a great resource. they should have some good suggestions for both positive and negative controls, which should be performed before you start monkeying around with titrating reagents (IMHO)
I hope you can solve your problem. that sounds quite frustrating
Before anything else: are you sure your primers are okay, meaning: have you tried to perform regular PCR on DNA? (Or is it too large with the intron).
Also: any way to control quality of your RNA? What's the size of your amplicon? SSII isn't too good, SSIII is way better (we go up to 3,8 kb with about 15-20 copies with this enzyme).
Thanks for your input I will look into Ambions website and try the PCR on DNA, I know the size i should be expecting for both DNA and RNA. I've been reading alot on RT-PCR and everyone always mentions SST-III I may try this if I'm still having problems.