RT-PCR machine/setup problems - (Mar/10/2006 )

I am a graduate student who is responsible for getting our lightcycler up and running. I have ran a handful of control reactions using the recommendations made by the manual. I am not getting any fluorence with any of the DNA control samples. We are on your second machine with the same negative results. I am thinking that it is something that I am doing wrong. I used fresh reagents and used sterile tips for the different runs. One thing that we haven't done is sterilize the capillaries. Do they have to be autoclaved? We just use them fresh from the sealed box.

Any suggestions would be of great help here since this is setting back my graduation date.

Sincerely,

Kevin Miller

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The capillaries are fine out of the box.

If you are absolutely sure that everything was right, you included all reagents (also added the enzyme to the mastermix before use, etc.) and you have cDNA in your reaction, than it might be the primers.

You could test your primers in conventional PCR to see if they work at all...

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Thanks for the idea. I ran the control kit reagents using conventional PCR. I ran two sets. One set was with SYBR green master mix and the other set was with our normal master mix. I ran three different dilutions of the control kit DNA. I only got one band and that was with the most concentrated sample with our "normal" master mix. The band was faint, but it was still a band. I kind of guessed on the times for the different cycles, but the temperatures where the same as for the RT-PCR protocol. It looks as though the primers and DNA are fine, so any other suggestions?

We do have a batch concentrated SYBR gold, would it work to add a diluted amount of SYBR gold and then run on the lightcycler? If so, how dilute would be recommended. I know that SYBR gold emits at a slightly different wavelenth, but at this point we are just trying to get something on the machine.


Thanks for any ideas.

Sincerely,

Kevin Miller

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