I am doing RT-PCR. All primers span more than one intron. I always prepare the non-RT reactions first and then RT reactions. Several thing always confuse me. 1) Sometimes I get the bands with correct size in some of the non-RT controls (preparation without thermoscriptase). But I never have problem with these two negative controls: water control from non-RT and water control during the PCR preparation. 2) I use beta-actin from mouse as an internal control. The same amount of total RNA (read at OD 260) is used for preparing cDNA in a 20 ul volume and 0.5 ul is used for subsequent PCR (also in 20 ul volume). All the PCR products are subjected to electrophoresis. The problem here is that sometimes the intensity of action varies so greatly: maybe 3-5 fold differences among samples. Another problem is in some cases even I get no band in actin control I still can amplify other genes from the same samples.
Can anyone explain these?
Any comments will be highly appreciated.
Edited by alex_osu3, 14 April 2004 - 08:30 AM.