Amplification in NTC and noRT controls - (Nov/12/2009 )
Dear All,
It has been 2 weeks now, and I'm growing more desperate. I have the same size band in my two negative controls (NTC and noRT) and my sample, which I want to show that this gene is not present. I used a positive control, that has about 1.5 times the amount of amplicon in the exact same position on the gel. Really weird. I have decontaminated everything. The product is over 100bp, so I don't think its a primer dimer effect, and it occurs within 25 cycles. The only thing I can think of is that the TE buffer I used to dilute the primers is contaminated. Any other ideas?
Thanks, C
charmspiet on Nov 12 2009, 05:33 PM said:
It has been 2 weeks now, and I'm growing more desperate. I have the same size band in my two negative controls (NTC and noRT) and my sample, which I want to show that this gene is not present. I used a positive control, that has about 1.5 times the amount of amplicon in the exact same position on the gel. Really weird. I have decontaminated everything. The product is over 100bp, so I don't think its a primer dimer effect, and it occurs within 25 cycles. The only thing I can think of is that the TE buffer I used to dilute the primers is contaminated. Any other ideas?
Thanks, C
Sounds like DNA contamination in your RNA. DNase treatment will help, but no matter how much you DNase treat, you won't get rid of all the DNA, just most of it. You may be able to dilute samples down so that the DNA contamination is negligible, but still have enough cDNA to detect.
fishdoc on Nov 12 2009, 07:16 PM said:
charmspiet on Nov 12 2009, 05:33 PM said:
It has been 2 weeks now, and I'm growing more desperate. I have the same size band in my two negative controls (NTC and noRT) and my sample, which I want to show that this gene is not present. I used a positive control, that has about 1.5 times the amount of amplicon in the exact same position on the gel. Really weird. I have decontaminated everything. The product is over 100bp, so I don't think its a primer dimer effect, and it occurs within 25 cycles. The only thing I can think of is that the TE buffer I used to dilute the primers is contaminated. Any other ideas?
Thanks, C
Sounds like DNA contamination in your RNA. DNase treatment will help, but no matter how much you DNase treat, you won't get rid of all the DNA, just most of it. You may be able to dilute samples down so that the DNA contamination is negligible, but still have enough cDNA to detect.
Thank you for your reply. But I was wondering, why then would there be amplification in my no template control (NTC)?
what about the water that you are using?? is it deionised DNA free water??!!
charmspiet on Nov 13 2009, 09:40 AM said:
Yeah, sorry about that. Completely missed that you did NTC, too. It's either in one of the reagents or it's getting picked up from the environment while setting up the reactions. Do you set up your reactions under a laminar flow hood? Do you set up your reactions where there is possible contamination of other PCR amplicon that could affect your results if picked up?
We set up all RT reactions and real time reactions under laminar flow. Prior to using the hood, we treat it with UV light and Virkon to destroy any DNA or other contamination that might be present.
fishdoc on Nov 14 2009, 06:51 AM said:
charmspiet on Nov 13 2009, 09:40 AM said:
Yeah, sorry about that. Completely missed that you did NTC, too. It's either in one of the reagents or it's getting picked up from the environment while setting up the reactions. Do you set up your reactions under a laminar flow hood? Do you set up your reactions where there is possible contamination of other PCR amplicon that could affect your results if picked up?
We set up all RT reactions and real time reactions under laminar flow. Prior to using the hood, we treat it with UV light and Virkon to destroy any DNA or other contamination that might be present.
Use the injection water or nuclease free water in your PCR reaction. you can get the vials from any pharmacy.
If the problem persists then make a fresh alpiqut of primers.