real time pcr amplification of non-template controls?

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bsengez
bsengez's picture
real time pcr amplification of non-template controls?

Hi,
We’re using SYBR green qPCR master mix from Fermentas to check the expression level of our transfected cell lines. I’m getting a signal in my template free negative control (nuclease free water instead of template) after 23th cycle. I also confirmed it on gel. I have changed supply of nuclease free water and purchased new primers and still get a signal. I couldn’t find what is wrong.
Any help would be appreciated.

R Bishop
R Bishop's picture
I had the same problem about

I had the same problem about off and on for years (ive done a ton of qPCR).  Although I can't account for the exact causes here's a checklist of what I changed to stop this from time to time.
 
1. Set up your reactions in another lab.  This worked for two primer sets.  I suppose we had so much DNA contaimination in our lab that the gene was on everything even the dust.
2. Pipetman - use fresh fllter tips for EVERY STEP - from resuspending primers to making you reactions. Clean your pipetman religiously and even dedicate a set to qPCR
3. Examine your surroundings and clean the surroundings within 20ft of your workbench.  Is the ventalation in your lab blowing from another scientists bench area? a bacterial incubator? etc.
4. Try a different batch of qPCR master SYBR mix.  One time it was totally contaiminated, I suspected a not so careful labmate.
5. order a different set of primers to the same gene (by this I mean new primer sequence).
6. Designate a tube rack for setting up reactions and never get it any where near finished qPCR reactions for any reason. If you do. Throw it away or never use it for qPCR again.
7. Use new diH2O for resuspending the primers when you reorder.  Made this mistake once, cost me a month.
Those were the most helpful.  Its tedious to be so careful, but it will pay off.  I'll try to thing of some more as the day goes along.
 
Rus

heehawmcduff
heehawmcduff's picture
 The ventilation issue is

 The ventilation issue is something I had a problem with - I found out that when the door was open in the room where I was doing the PCR, it dramatically altered the flow of air around the class 2 tissue culture hood.  It's a small wonder that my cell lines didn't get contaminated.

Ivan Delgado
Ivan Delgado's picture
While I agree 100% with Rusty

While I agree 100% with Rusty's recommendations, if you get amplification at cycle 23 on your NTC you definitely have contamination problems that will not be solved by setting up your assays in a separate room or checking the ventilation in your laboratory. To get amplification at cycle 23 you must have a lot of starting template in your assays and you cannot introduce that level of contamination through aerosols. Alternatively this could be an issue of assay design, but I assume you are running a standard curve and see amplification at earlier cycles (before cycle 23) when you add template to your assay. 
My recommendation is to run, side by side, your assay and another assay you know works well (no amplification in the NTC). Maybe someone you know has an assay they use frequently? If both assays show amplification in the NTC then it is a contamination problem. If only your assay shows contamination, then it is possibly an issue with your assay design. 
Good luck