PCR is Polymerase Chain Reaction which is a commonly used technique in molecular biology experiments to amplify a single DNA molecule.

I have been working on it for months and can’t eliminate contamination even until now. I want to share with you my experience which may help in the future.

My experience is divided into two parts. First, nested PCR; second, amplify ChIP samples.

Nested PCR Contamination in the Second Round

If you know what PCR is, you can Google and find out what nested PCR is. It is two rounds of PCR with a pair of second round primers within the first round PCR product. So that you can get a shorter second round PCR product than the first round.

Everything was fine at first. But when I used a new pair of primers, I got contamination of the first round product (~300bp) in the second round, which product should be ~200bp. Then I did everyone would do when facing PCR contamination.

1. Repeat it again, and the result is the same;
2. Change the gloves, labcoat, PCR water, new reagents, new tips, still the same;
3. Perform the PCR in a PCR hood, the result is worse: more samples were contaminated

Then I asked my boss and he advised me to diluted the first round primers. I did it and found that when I diluted the first round primers to 1/20, I got no signal in the second round amplification without adding the second round primers. The specificity of the first round primers must be very high that it competed efficiently with the second round primers in a very low concentration.

That is the first story.

Contamination in the ChIP Samples

When I performed PCR to amplify the ChIP samples, I got consistent contamination in the NTC (negative control), which had no template adding. My target product was about 200bp, but the contamination was a gradient with the largest product about 2kb! When I first time saw it, I even thought it a marker!!

I tried all the methods I used above and even change to a new reagent from other company and asked another person to help me perform it, but the contamination is consistent. Then I thought it might be the problem of the original primers, so I change to a new pair of primers. However, the contamination still existed.

Accidently I wrongly added 500uM dNTP rather than 250uM (that is 1ul 10mM dNTP in 20ul system rather than 0.5ul), I got the NTC clean! And the result was repeatable.

Now I am still amplifying the ChIP samples using the same methods, and don’t know where the contamination is from.

Now the story is at one of its end, and I want to tell you that if you are not a newbie of PCR, when you meet the contamination, it always does not from the water, reagent, gloves, circumstances, pipet, tips, and person. Most commonly, it is from primers, and then some unknown reasons.

Trust yourself. You are not the source and reason of contamination!!!