Troubleshoot PCR, Product band missing, internal control is present - (Apr/11/2011 )

Hello everybody,

I am new to research and recently learned to do Reverse Transcription, PCR and Gel Electrophoresis. Since the past two experiments my results have not been very good.

When I run my agarose gel (2%), my internal control (G3PDH) shows up perfectly fine but my target (iNOS) does not show up at all. When using the Nanodrop to check the RNA quality report, the data was excellent - no protein contamination, etc.

Based on this, if my internal control shows up on my gel, can I conclude that my PCR, and Reverse transcription were fine and there are other things wrong? Could it be possible that primers are not good from repeated thawing?

I would greatly appreciate if someone can tell me their opinion because this is the second time I've done this experiment (time course) and first I thought it was me due to my inexperience but now it seems there is something else at work.

Thank you

Samanouske,

You are right about the PCR and reverse transcription. There can be many reasons why your expected PCR product is not seen.

Like:
1. Primers are no longer good.
2. Primers are good but the reaction conditions are suitable for good amplification of the PCR product
3. The reaction conditions are also fine, but product formed is not enough to be seen on the gel.
4. Primers are incorrect and do not bind to the target DNA.
etc.

You would have to give us some information, like PCR conditions, melting temperatures of the primers, age of the primers, (if anybody else has used them before), before we can locate the problem areas.