contamination in real-time PCR - (Apr/01/2005 )

Ah... the old contamination chestnut...

If the product in your no-template controls is amplified by your gene-specific primers, then the problem is likely to be contamination with your PCR product - follow instructions in the posts above to resolve this,

If you're using 18S rRNA as a housekeeping gene, and your unexpected product is being amplified from 18S primers, don't forget that these primers are generally 'universal' - so they will amplify the 18S gene from almost *any* eukaryote. Fungi are the usual suspect here. If you're using 18S primers you should autoclave and UV-irradiate your PCR tubes / plates before use. If you don't have a UV-crosslinker, you can invert a transilluminator and irradiate the (open) tubes / caps in racks underneath for 10-15 minutes or so.

good luck,

Del.

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I did not know what type of dye you have used, SYBR Green or Taqman probe? If you are using SYBR Green, you may need to worry about primer dimers. If you are using the taqman probe, it dose not make sense primer dimers will show the amplification, because no amplicon will be generated by primer dimers. Amplification shows up only when the probes are broken down during amplicons synthesis.
I am using ABI Prism 7000 machine I noticed that sometimes the noise may give me wrong report when threshold line was set too low. Please check the threshold line settings and amplification curve. Change the threshold line settings may also change Ct value a lot.

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This paper has some interesting studies on using DNAse, UV, restriction enzymes, and 8-MOP to overcome contamination:

http://www.pubmedcentral.nih.gov/articlere...cgi?artid=86577

Has anyone here ever tried 8-MOP or DNAse treatment? The paper above describes a significant loss of sensitivity when using it 8-MOP, but very little using DNAse in combination with AvaI.

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Hi flash boy,
maybe you should consider to use UNG for carry-over prevention and dUTP in your dNTP mix. Furthermore, did you check if your primers are forming dimers? Did you do a testrun of this PCR-product on an analytical agarose gel?
I hope this might give you some hints.
Cheers,

ophris

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This problem is probably due to contamination of amplified products in the air from previous runs using the same primers. If your room has no windows that can be opened and is central air conditioned, I bet this is it. All you need to do is to open the windows and let the air circulate. We have been using a huge fan to blow the lab once in a while.

Also, never open a real-time PCR tube in the lab where you prepare your real-time PCR reactions. If you run an agerose gel to check your real-time PCR amplification, you contaminate the lab. It is very hard to get rid of the air contamination - it usually takes months before your negative control shows negative again.

Jing

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hi guys,

just to let you know that the 'top guy' from biorad told me this was normal and that it was not to be taken too seriously!!!!

have run through a gel and can't see anything

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Seriously, think about what Bio-Rad is saying...its ok that non-specific (supposedly, but kinda' weird because its coming up in a TaqMan assay) amplification is occuring after 35 cycles of PCR. I would think about conventional PCR, is the NTC (PCR negative) reaction come up weakly positive after 35 cycles, not usually. It is important to remember that your TaqMan probe is somehow being degraded in a log fashion in order to produce the observed fluorescence data you're seeing late on your amplification plots. Thus, this is NOT probe degradation. This is probably due to contamination, as you said, albeit at a low level. What is your target for amplification? Is it a human target, such as GAPDH? What conditions are you using to set up your reaction? I would suggest turning over your workspace with cleaning, because if somehow a previously-amplified product ended up in your hood, HEPA filter, etc, there are potentially billions of copies blowing around in the hood during PCR preparation, or even DNA/RNA extraction.

C

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sorry, there must be some confusion, this is SYBR green real-time which gives me a late signal in my neg controls

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sorry, there must be some confusion, this is SYBR green real-time which gives me a late signal in my neg controls
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My apologies, I should have read the previous posts more thoroughly. Non-specific SYBR green amplification is a weird thing, and as you know, any DNA duplex formation will result in SYBR green detection. It is either primer-dimerization or of some sort or contamination. If you are interested in detecting low copy numbers (i.e. close to where you late signal is coming up), the SYBR green methodology using this primer set may not be the best option. Is it possible to just add a TaqMan probe to the primer set you already have? This will almost assuredly cure the late SYBR green amplification. As other have spoken about before, contamination with your target DNA, either from experimental samples (less likely) or with post-run PCR products (more likely) will not be cured by converting to TaqMan. Good luck...

C

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hello,
Please checkout the properties of your primers. Does it contain primer-dimer? Strong primer-dimer will bind among themselves during PCR amplification. As a result you will observe an amplification curve in your real-time.

Thank you.

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