PCR Troubleshooting: The Template DNA

The DNA in a PCR reaction comprises two types:

• the target sequence to be amplified
• the non-target DNA (also called the "burden" DNA
The amount of total DNA in a PCR has a marked effect on the outcome of a PCR procedure.
Using too much total DNA results in packed DNA in the confined space of the reaction vessel and
can lead to false priming and even poor DNA synthesis due to the obstructed diffusion of large
Taq polymerase molecules. However the ratio of target DNA to burden DNA is also important.
The concentration of the target DNA should be balanced with the number of cycles in the
reaction. Using an elevated concentration of the target combined with the normal, or higher than
normal, number of cycles can cause the accelerated accumulation of nonspecific products. The
accumulation of nonspecific products is often observed in a reamplification PCR, when the high
initial concentration of the PCR fragment is accompanied by a high number of cycles. Reducing
the number of cycles may help. However, low concentrations of primer, target, Taq, and
nucleotides are recommended as these generally ensure cleaner product and lower background.
Problems also occur when the ratio of the target DNA to the burden DNA is very low, for example
the amplification of a 500 bp fragment from the human genome (1 to 6 x 106). A better ratio is
between 1:1 and 1:1 x 104. A ratio of 1:1 is achieved in a reamplification reaction and a ratio of
about 104 is achieved when amplifying from the Escherichia coli genome.

When the total amount of the DNA in a PCR reaction is extremely small, there is an
increased likelihood of its loss owing to any conceivable cause (clotting, adsorption, chemical or
enzymatic degradation). Furthermore, a small amount of target DNA leads to an increased risk
from contaminating DNA from impurities on anything that can come into contact with the DNA
solution. In this respect, both the DNA diluent, the dust floating in the air, exhalations and even
particles of skin or hair from your body should not be disregarded, as these can carry both the
DNA and the DNA-degrading substances. Nucleases are probably as the major cause of DNA
degradation in a PCR procedure. They are abundant on the surface of the human skin and can
be present everywhere else too. Mild autoclaving of the DNA diluent and everything that comes in
regular, occasional, or accidental contact with buffers and solutions will destroy both the
nucleases and comtaminating DNA. If you suspect problems of this nature, wear gloves, a
surgeon's cap, and a face mask. Also, wash the working space with an oxidizing substance such
as (6% H202).

PCR Troubleshooting: Inadequate dNTPs

An incorrect concentration of deoxynucleotidetriphosphates (dNTPs) can cause problems

for the PCR procedure. The usual dNTP concentration is between 40μM and 200μM of EACH of
the four dNTPs. Excessive dNTP concentrations can inhibit the PCR preventing the formation of
product. However, concentrations up to 400 μM each dNTP have been reported to work
adequately. Low primer, target, Taq, and dNTP concentrations are preferable as these generally
ensure cleaner product and lower background. For longer PCR-fragments a higher
deoxynucleotidetriphosphate concentration may be required. A large change in the dNTP
concentration may require a corresponding change in the concentration of MgCl2.
Suboptimal concentration of nucleotides can cause incomplete primer elongation or premature
termination of DNA synthesis during the elongation step of the PCR cycle.

PCR Troubleshooting: Primer Concentration

The recommended primer concentration for PCR is between 0.1μM and 1μM of each
primer. The use of higher concentrations of primers can have the following effects:
If the primers are capable of forming dimers, raising their concentration only results in the
creation of primer-dimers and does not improve the amplification of the desired PCR product.
Primer-derived oligomers will possibly contaminate the reaction.
If the primers do not form primer-dimers, it is likely that raising the primer concentration will lead
to non-specific primer binding and the creation of spurious, undesirable PCR products.
Raising the primer concentration does not therefore cause an increase in the effective
concentration of the primers. Low primer concentration generally ensures cleaner product and
lower background.
However, to amplify short PCR target sequences, careful calculation of the optimum primer
concentration is required. For example, if the target fragment length is 100bp, a greater number
of PCR product molecules is required to provide a specified amount of amplified DNA (in
nanograms) than for a larger target fragment. In order to generate the required number of PCR
product molecules, a greater number of primers may be needed. Therefore, concentration of
primers higher than 1μM may be necessary, and desirable, for short target sequences.

PCR Troubleshooting: Taq Concentration

In a PCR experiment approximately 1 unit of the Taq enzyme should be used for a 25μl
reaction. Suboptimal concentration of the Taq enzyme can cause incomplete primer elongation or
premature termination of the PCR product synthesis during the elongation step of a PCR cycle.
Too much Taq will result in an excessive background of unwanted DNA fragments (a smear on a
gel) while a huge excess may cause the reaction to fail with no product being detected. A Taq
concentration of 1 unit per 25μl reaction ensures a cleaner product and lower background.

PCR Troubleshooting: Mg Concentration

Magnesium is a required cofactor for thermostable DNA polymerases. Mg2+ in the PCR
mixture stabilizes dsDNA and raises the Tm. Mg2+ concentration therefore is an important for
controlling the specificity of the reaction. A low Mg2+ concentration requires more stringent base
pairing in the annealing step. Too few Mg2+ ions result in a low yield of PCR product; too many
Mg2+ ions increase the yield of non-specific products and promote misincorporation.
Insufficient Mg2+ concentration in a PCR mixture can causes failure of the reaction. Excess
magnesium (or the presence of manganese) will cause the fidelity of DNA polymerases to be
reduced and may cause the generation of unwanted products. On a gel this can appear as a
ladder or smear. The MgCl2 concentration should normally be between 1mM and 4mM. Since
dNTPs sequester Mg2+ ions, a major change in the dNTP concentration in a rection would
require a change in the concentration of MgCl2. Similarly, changing the KCl-based buffer
concentration or any other component of the PCR mix may require adjustment of the Mg2+
concentration in the reaction mixture.

PCR Troubleshooting: KCl Concentration

Potassium chloride (KCl) is normally used in a PCR amplification at a final concentration

of 50mM. To improve the PCR amplification of DNA fragments, especially fragments in the size
range 100bp to 1000bp, a KCl concentration of between 70mM and 100mM is sometimes
recommended. For the amplification of longer products a lower salt concentration appears to be
better. But the PCR amplification of short products works better at higher salt concentrations. This
is probably because an increase in salt concentration permits shorter DNA molecules to denature
preferentially to longer DNA molecules. Shorter molecules are therefore amplified better at higher
salt concentration. It should be remembered however that a salt concentration above 50mM can
inhibit the Taq polymerase.
If you are finding unwanted, long, non-specific products an increase in KCl concentration may
reduce the appearance of these products. Similarly, to get rid of short, non-specific products you
can decrease the KCl concentration to about 35 or 40mM. In either case do not change the
MgCl2 concentration. To improve the yield of a product you can try adjusting the KCl
concentration: increase it for a desired product less than 1000bp; lower it for a desired product
greater than 1000bp.
Troubleshooting discussion is based on the PCR protocol as described in the table below. All
reactions are run for 30 cycles.

COMPONENT VOLUME FINAL CONCENTRATION

1.autoclaved ultra-filtered water (pH 7.0) 20.7µL -

2.10x PCR Buffer* 2.5µL 1x

3.dNTPs mix (25 mM each nucleotide) 0.2µL 200 µM (each nucleotide)

4.primer mix (25 pmoles/µL each primer) 0.4µL 0.4 µM (each primer)

5.Taq DNA polymerase (native enzyme) 0.2µL 1 Unit/25 µL

6.genomic DNA template (100 ng/µL) 1.0µL 100 ng/25 µL

* The 10x PCR buffer contains: 500 mM KCl; 100 mM Tris-HCl (pH 8.3); 15 mM MgCl2 (the final
concentrations of these ingredients in the PCR mix are: 50 mM KCl; 10 mM Tris-HCl; 1.5 mM
MgCl2).

QUESTIONS

1. I get (many) longer unspecific products. What can I do?

• Decrease annealing time
• Increase annealing temperature
• Decrease extension time
• Decrease extension temperature to 62-68º C
• Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-
2mM.
• Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
• Take less primer
• Take less DNA template
• Take less Taq polymerase

If none of the above works: check the primer for repetitive sequences (BLAST align the sequence
with the databases) and change the primer(s)
Combine some/all of the above

2. I get (many) shorter unspecific products. What can I do?

• Increase annealing temperature
• Increase annealing time
• Increase extension time
• Increase extension temperature to 74-78º C
• Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-
2mM
• Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant
• Take less primer
• Take less DNA template
• Take less Taq polymerase
If none of the above works: check the primer for repetitive sequences (BLAST align the sequence
with the databases) and change the primer(s)
Combine some/all of the above
3. Reaction was working before, but now I can't get any product.
• Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc)
• Change the dNTP solution (very sensitive to cycles of thawing and freezing, especially in
multiplex PCR)
• If you just bought new primers, check for their reliability (bad primer synthesis ?)
• Increase primer amount
• Increase template amount
• Decrease annealing temperature by 6-10º C and check if you get any product. If you
don't, check all your PCR ingredients. If you do get products (including unspecific ones)
reaction conditions as described above.
• Combine some/all of the above

4. My PCR product is weak. Is there a way to increase the yield?

• Gradually decrease the annealing temperature to the lowest possible.
• Increase the amount of PCR primer
• Increase the amount of DNA template
• Increase the amount of Taq polymerase
• Change buffer (KCl) concentration (higher if product is lower than 1000bp or lower if
product is higher than 1000bp)
• Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5%
(v/v, final concentration) DMSO or glycerol.
• Check primer sequences for mismatches and/or increase the primer length by 5
nucleotides
• Combine some/all of the above

5. My two primers have very different melting temperatures (Tm) but I cannot change their
locus. What can I do to improve PCR amplification?
• An easy solution is to increase the length of the primer with low Tm. If you need to keep
the size of the product constant, add a few bases at the 3' end. If size is not a concern,
add a few bases at either the 3' or the 5' end of that primer.

6. I have a number of primer pairs I would like to use together. Can I run a multiplex PCR
with them?. How?
• Very likely, yes.
• Try amplify all loci seaprately using the same PCR program. If one of the primer pairs
yields unspecific products, keep the cycling conditions constant and change other
parameters as mentioned above (#1 and #2).
• Mix equimolar amounts of primers and run the multiplex reaction either in the same
cycling conditions or by decreasing only the annealing temperature by 4º C.
• If some of the loci are weak or not amplified, read below !!

7. How many loci can I amplify in multiplex PCR at the same time?
• Difficult to say. The author has routinely amplified from 2 to 14 loci.
• Literature describes up to 25 loci or so.

8. One or a few loci in my multiplex reaction are very weak or invisible. How can amplify
them?
• The first choice should be increasing the amount of primer for the "weak" loci at the same
time with decreasing the amount of primer for all loci that can be amplified. The balance
between these amounts is more important than the absolute values used !!.
• Check primer sequences for primer-primer interactions

9. Short PCR products in my multiplex reaction are weak. How can I improve their yield?
 • Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM
• Decrease denaturing time
• Decrease annealing time and temperature
• Decrease extension time and temperature
• Increase amount of primers for the "weak" loci while decreasing the amount for the
"strong" loci.
• Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5%
(v/v, final concentration) DMSO or glycerol
• Combine some/all of the above

10. Longer PCR products in my multiplex reaction are weak. How can I improve their
yield?
• Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-
2mM
• Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
• Increase denaturing time
• Increase annealing time
• Decrease annealing temperature
• Increase extension time and temperature
• Increase amount of primers for the "weak" loci while decreasing the amount for the
"strong" loci
• Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5%
(v/v, final concentration) DMSO or glycerol
• Combine some/all of the above

11. All products in my multiplex reaction are weak. How can I improve the yield?
• Decrease annealing temperature in small steps (2º C)
• Decrease extension temperature to 62-68º C
• Increase extension time
• Increase template concentration
• Increase overall primer concentration
• Adjust Taq polymerase concentration
• Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM
• Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant.
• Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5%
(v/v, final concentration) DMSO or glycerol
• Combine some/all of the above

12. Unspecific products appear in my multiplex reaction. Can I get rid of them somehow?
• If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at 1.5-2mM
• If short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration at 1.5-
2mM
• Gradually increase the annealing temperature
• Decrease amount of template
• Decrease amount of primer
• Decrease amount of enzyme
• Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant
• Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5%
(v/v, final concentration) DMSO or glycerol
• If nothing works: run PCR reactions for each (multiplexed) locus individually, using an
annealing temperature lower than usual. Compare the unspecific products for each locus
tested with the unspecific products seen when running the multiplex PCR. This may
indicate which primer pair yields the unspecific products in the multiplex reaction.
• Combine some/all of the above
• (Note: primer-primer interactions in multiplex PCR are usually translated into lack of
some amplification products rather than the appearance of unspecific products)