A couple of (RT-PCR) cowboy questions - I feel the need, the need for speed (May/07/2008 )
I am in the process of collecting hundreds of samples for RT-PCR and I am looking for ways to minimise the time it's going to take without jeopardising my results.
My first question is, can I re-use my TBE running buffer for a few gels? I'm sure the lab I did my undergrad project in did this but people in my current lab don't seem to (but then most of them are running tiny 18 sample gels not the 80 well bad boys that I use).
My second question is, are RT negatives necessary in every PCR? A friend of mine told me that you only have to do them once (i.e. in the housekeeping gene) to check the purity of your RNA. Also you should do them if your primers are not in different exons. This makes sense to me but it feels like it might be a bad idea and cutting one too many corners.
Any advice/opinions will be greatly appreciated.
Helen
My first question is, can I re-use my TBE running buffer for a few gels? I'm sure the lab I did my undergrad project in did this but people in my current lab don't seem to (but then most of them are running tiny 18 sample gels not the 80 well bad boys that I use).
My second question is, are RT negatives necessary in every PCR? A friend of mine told me that you only have to do them once (i.e. in the housekeeping gene) to check the purity of your RNA. Also you should do them if your primers are not in different exons. This makes sense to me but it feels like it might be a bad idea and cutting one too many corners.
Any advice/opinions will be greatly appreciated.
HelenIMHO, the answers are yes, and YES:
Depending how long/fast you're running your gels, you should be able to rerun your buffer at least a couple times. (but I wouldn't do it if you're running at really high voltage or for several hours.)
As far as the RT controls, I would say that a negative RT control needs to be run for every RT (positive) sample you have. Also, always run a "no template" control for every primer set.
Hope this helps
by the way, I loved the topic name!
Thanks, I kind of thought that was probably the answer. I think I will try using the buffer two or three times and see how it goes. In my heart I knew that I should run RT negatives in every PCR I was just hoping I might get out of it!