Ten Things That Can Kill Your PCR

by Peter Frame

A blank PCR gel has got to be one of the most aggravating things about

molecular biology. We've all had PCRs work one day, then fail inexplicably

the next. And we've all banged our heads against a wall trying to figure out

what went wrong. If your gels are turning up blank, check out this list of

things that can kill your PCR -- and get your experiments working!

Nowhere is the old adage "an ounce of prevention is worth a pound of

cure" more applicable than to PCR. Depending on your level of PCR

expertise, some of these hints may seem obvious. But how many times

have you kicked yourself because you overlooked the obvious? Whether

you're an expert or a novice, any of these may be just what you're

searching for to get your PCR working.

1.Too much dNTP, or degraded dNTP. Too much dNTP can actually

inhibit your PCR reaction. Between 40 - 200 uM is the optimal range.

Also, dNTPs are sensitive to repeated freeze-thaw cycles. Make

small aliquots when you get a fresh batch, and turn over your stock

frequently since dNTPs frozen at -20 C will eventually go bad.

2.Not mixing MgCl2. Magnesium chloride solutions form a

concentration gradient when frozen and need to be vortexed prior to

use (1).

3.Wrong MgCl2 concentration. Every PCR reaction has an optimal

MgCl2 concentration range, usually between 1 - 4 mM. Mg2+ ions

form complexes with dNTPs and can also act as a co-factor for

polymerases, so you'll need to try several conditions to optimize your

concentration.

4.Inhibitors in your reaction. Make sure you know how you got your

source DNA. Chloroform, phenol, EDTA, ionic detergents (SDS and

Sarkosyl), xylene cyanol, bromophenol blue and ethanol -- among

many other things -- can inhibit PCR. An extra 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.

5.Poor quality mineral oil. Lower-grade preparations may contain

nucleases that can kill your PCR. Also, avoid autoclaving your mineral

oil if possible. Exposure to high heat may cause reaction-inhibiting

hydrocarbons to form. Similarly, do not irradiate mineral oil with UV

for long periods (2).

6.Too much enzyme. Excess enzyme in your PCR can lead to

smearing of PCR products. Most people seem to use 0.5 ul of their

stock Taq per reaction, but that may contain way more than

necessary for your particular reaction.

7.Wrong primer concentration. If you have too little primer you

won't see any product. Too much primer and you may get primer

dimerization and not enough amplification. Stay within 0.1 - 1.0 uM

of primer.

8.Wrong PCR program. Make sure the program you selected on your

PCR machine is actually the one you want! It only takes a slip of a

finger, or some klutz, to alter your personal program on a common

PCR machine. Check your program while it's cycling to make sure it's

what you wanted.

9.Excess or insufficient template. Too much template can inhibit

PCR by binding all the primers. Too little template, and amplification

may not be detectable. For 25 - 30 cycles, 104 copies of the target

sequence are sufficient.

10.Poor primer design. While primer design can seem like a black art,

avoid obvious errors like self-complementarity, complementarity

between paired primers, or excessively long oligos (>30 bp). Often,

making a new primer next to a suspect one can solve the problem

and can be faster and cheaper than trying numerous variations in

reaction conditions.

Of course, this list is by no means comprehensive. Like any experiment,

many things can go wrong. If you have anything you'd like to add, we'd

love to know about it. Drop us an email at feedback@biowire.com. We'll

compile another set of PCR Do's and Don'ts and publish them in the future.

In the meantime, may all your PCRs work the very first time!

References:

1. http://www.md.ucl.ac.be/didac/sbim2520/pcr.html

2. Dohner DE, Dehner MS, Gelb LD. Inhibition of PCR by mineral oil exposed to UV

irradiation for prolonged periods. Biotechniques 1995; 18(6): 964-7.