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Why PCR goes wrong …

Why PCR goes wrong …

Are you doing PCR? Has your experiment failed and left you wondering why? Do you need a short checklist of essentials before every experiment? If the answer to at least one of these questions is a “Yes” then this article is for you!

If you are doing PCR, then you know that there is no such thing as routine PCR. What we mean by this is that even with the simplest PCR reaction, there are so many steps that could go wrong.

Here are a few simple factors, which may affect or make your PCR experiments fail if not done effectively:

1.      Not mixing MgCl₂ – Magnesium chloride solutions form a concentration gradient when frozen and need to be vortexed prior to use.

2.      Wrong MgCl concentration – Every PCR reaction has an optimal MgCl₂ concentration range, usually between 1 – 4mM. Mg²+ ions form complexes with dNTPs and can also act as a co-factor for polymerases. Hence you will need to try several conditions to optimise your concentration.

3.      Inhibitors in your reaction – Ensuring you know how you got your source DNA, is an essential factor for your reaction. Chloroform, phenol, ionic detergents, xylene cyanol, bromophenol blue and ethanol, can inhibit PCR. An additional clean-up step on your template may do the trick. Also, certain polymerases can be more susceptible to certain substances, so be sure to check your polymerase for possible inhibitors.

4.      Wrong primer concentration – Finding the right amount of primer can be tricky for many new PCR starters – if you don’t have enough primer you won’t see any product! On the other hand too much primer and you may get primer dimerisation and not enough amplification. Therefore, we recommend you stay within a final concentration range of 0.1 – 1.0 µM for each primer.

5.      Wrong cycling conditions – This is where it all goes wrong. First, make sure the cycling conditions you selected on your PCR machine are actually the correct ones for the specific reagent and experiment you are currently running!

Also, be aware that there are external factors which may affect your PCR runs. It only takes a slip of a finger, or a clumsy person, to alter your personal programme on a common PCR machine. Check your programme while it’s cycling to make sure it’s what you wanted.

Our Appleton PCR reagents range covers a broad spectrum of polymerases and ready mixes to minimise errors which could occur in your experiments, and are flexible to deal with complex DNA targets. The Appleton reagents have a buffer system with pre-added dNTPs and MgCl₂. With our mixes you can be confident that the MgCl₂ and dNTP concentrations have been pre-optimised for the protocol you are using. This simple extra means our product eliminates your concern about the MgCl₂ aspect of the PCR run. There’s also no need to buy separate dNTPs – our ready-mixes come with these already added. We all like saving on costs, don’t we?

Additionally, our reagents come with carefully written protocols containing the cycling conditions and the steps you need to take when preparing for your experiment.

Finally, if you would like to clean up that source DNA, we have just the kit for you. Our Appleton Gel & PCR clean-up kit not only cleans up DNA after gel electrophoresis and PCR, but is flexible enough to clean up contaminants from all kinds of enzymatic reactions, e.g. restriction digests and ligations.

Let’s make your PCR easy and stress-free…click here to browse our range!