Mark Fry <wmfry at morgan.ucs.mun.ca> wrote:
>I'm having a problem with my RT-PCR reaction. I have done the RT three
>times and still no PCR products.
>I start with about 2ug total RNA (in 16ul depc H2O) and I know this is
>good quality stuff.Heat the RNA to 80 C for 3 min, place on ice for 3 min.
>I add (in order, on ice) 1.5 ul dNTP (10 mM each), 1 ul of 100 ng/ul oligo
>dT adaptor [primer 34 mer, 17-18 of which are dT), 6 ul 5X buffer (gibco),
>3 ul 0.1M DTT (gibco), 2.5 ul of superscript MMLV (Gibco). Incubate at 37
>C for 1 hour, and use as template in PCR.
>I run the PCR reaction along with a control template of a clone that
>gives the right product, so I know the primers work fine.
>I have also done this RT-PCR before, and it worked fine.
>Is it wise to treat the cDNA with RNase H or RNase A? Is there
>anything else I should Do?
Grin, One important piece of information that I found lacking in your
protocol and explanation was whether the mRNA in question is actually
expressed in your RNA prep. You have one decent control in your
protocol but perhaps you may want to add another, an RT-PCR using
primers for a known expressed mRNA. In this manner you could exclude
your RT-PCR methodology from your problem. If your still having
trouble feel free to email me or repost here and I will post the
RT-PCR method I am using.
--Have you hugged your DNA today?