RT-PCR NTC- substitute DNA with water? - or just end up with a smaller rxn volume? (Apr/12/2007 )
When making up a RT-PCR NTC (negative control, no DNA), do you add water instead of the DNA volume you're supposed to add?
I've been running PCR rxns since september and all my controls show a product (see previous thread), but I havent been adding anything to replace the DNA volume to the control mix, so it's always less by 5ul (rxn vol. of 20ul)--could this be my problem?
in PCR the concentration of all reagents is important - Primer, MgCl, buffer, and polymerase. Therefore it is important to use "real" water controls, i.e. add the adequate amount of water to the reaction mix.
I don't know if your problems arise only from that, but I would really be strict with concentrations when dealing with real-time PCR.
Hope next experiments will run better for you,