RT-PCR - necessary to run electrophoresis gels? - (Jul/24/2007 )
I am setting up a protein lab to run RT-PCR. There is some equipment here but no gel electrophoresis equipment and no agarose. Is it necessary to check at different stages of the process and after real time quantitative pcr by running a gel? Are there ways to do this without using EtBr? Also, any other tips you have will be appreciated. Thanks in advance!
Hi MTS,
if you setup a real-time RT PCR, checking your products on a gel is necessary, as the melting curves can always trick you. So you probably have to set up everything for gel electrophoresis. You can use other agents then EtBr, e.g. SYBR green. This is less toxic but more expensive.
It may also be helpful to have electrophoresis at hand to check RNA quality.
Krümel
if you setup a real-time RT PCR, checking your products on a gel is necessary, as the melting curves can always trick you. So you probably have to set up everything for gel electrophoresis. You can use other agents then EtBr, e.g. SYBR green. This is less toxic but more expensive.
It may also be helpful to have electrophoresis at hand to check RNA quality.
Krümel
hi
can you tell me how to check PCR products from real time with SYBR green chemistry? because i have experienced that nothing is seen on the gel.
Never had a problem. For dsDNA simply stain the gel in SYBRgreen I solution after the electrophoresis.
If you are using established, well set-up primer-probe systems, it is not necessary to check it on a gel. But while designing new primers/probes I routinely do...