EMERGENCY: ovine GAPDH NTC contamination - (Feb/14/2006 )

EMERGENCY. I am constantly having ovine GAPDH NTC contamination in my lab recently (has happened to a number of my runs).

The Ct for the contamination peak is around 33, and the dissociation curve showed it is a PCR product (80C) not a primer dimer. The other NTCs with testing genes are negative, hence i don't think this is a template cDNA contamination.

We have bleached our pipette, tip box, racks; used new aliquote of SyberGreen, new aliquote of water, and change gloves as much as we can. We even stop using airconditioning when set up plate to prevent the possible contamination in the air.

The other girl works at the other side of lab does not have any contamination.

We have the QPCR machine inside the lab, where we set up our plate.


DESPERATELY NEE HELP & ADVICES.

at Ct of 33 it is as good as not present. I am no expert of RT-PCR. My thoughts. I am sure others can throw more light on this.

I have got the same situation as u...... and now also seek for the other help and I have also seen in many forum concerning about this problem... But no one get the real reason behind except the contamination issue, but u know it is not the point as I also change the stuff as much as I can

Once I've worked on a lab and they had the same problem, other PCR's with different primers din't have this problem. After a long time of searching they found that the primers in stockt solution were contaminated. This could have happend during desolving the primers as a stock solution. They found out when they ordered new primers. I don't know if that's the case with you, but it's an idea.
Or maybe a buffer you used is contaminated.

Good luck.

Nieky

Load your realtime product on a standard EtBr-Agarose gel and check whether the NTC has a band (in right product size) or not. Because it could be that those are only primer dimers...

Do you have different pipettes and lab-bench for PCR setup and PCR products? That is highly recommended! You should also add the primermix into the NCT-well first, then add it to the wells containing template...and change the pipet-tip (filtered) for every well.
Good luck!

I agree with Nieky...the specificity suggests a problem with the primers

Once I've worked on a lab and they had the same problem, other PCR's with different primers din't have this problem. After a long time of searching they found that the primers in stockt solution were contaminated. This could have happend during desolving the primers as a stock solution. They found out when they ordered new primers. I don't know if that's the case with you, but it's an idea.
Or maybe a buffer you used is contaminated.

Good luck.

Nieky