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.

For example. For the amplification of longer products a lower salt concentration appears to be better. concentration of primers higher than 1μM may be necessary. 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: Taq Concentration In a PCR experiment approximately 1 unit of the Taq enzyme should be used for a 25μl reaction. lower it for a desired product greater than 1000bp. Therefore. long. On a gel this can appear as a ladder or smear. a greater number of primers may be needed. for short target sequences. A Taq concentration of 1 unit per 25μl reaction ensures a cleaner product and lower background. Since dNTPs sequester Mg2+ ions. it is likely that raising the primer concentration will lead to non-specific primer binding and the creation of spurious. The MgCl2 concentration should normally be between 1mM and 4mM. Raising the primer concentration does not therefore cause an increase in the effective concentration of the primers. and desirable. However. undesirable PCR products. But the PCR amplification of short products works better at higher salt concentrations. to get rid of short. To improve the yield of a product you can try adjusting the KCl concentration: increase it for a desired product less than 1000bp. This is probably because an increase in salt concentration permits shorter DNA molecules to denature preferentially to longer DNA molecules. a major change in the dNTP concentration in a rection would require a change in the concentration of MgCl2. Insufficient Mg2+ concentration in a PCR mixture can causes failure of the reaction. PCR Troubleshooting: KCl Concentration Potassium chloride (KCl) is normally used in a PCR amplification at a final concentration of 50mM. careful calculation of the optimum primer concentration is required. a KCl concentration of between 70mM and 100mM is sometimes recommended. Similarly. If you are finding unwanted.If the primers do not form primer-dimers. It should be remembered however that a salt concentration above 50mM can inhibit the Taq polymerase. too many Mg2+ ions increase the yield of non-specific products and promote misincorporation. 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. Similarly. Shorter molecules are therefore amplified better at higher salt concentration. especially fragments in the size range 100bp to 1000bp. Too few Mg2+ ions result in a low yield of PCR product. A low Mg2+ concentration requires more stringent base pairing in the annealing step. non-specific products you can decrease the KCl concentration to about 35 or 40mM. non-specific products an increase in KCl concentration may reduce the appearance of these products. PCR Troubleshooting: Mg Concentration Magnesium is a required cofactor for thermostable DNA polymerases. to amplify short PCR target sequences. 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. Mg2+ in the PCR mixture stabilizes dsDNA and raises the Tm. Low primer concentration generally ensures cleaner product and lower background. In either case do not change the MgCl2 concentration. 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. 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. if the target fragment length is 100bp. Mg2+ concentration therefore is an important for controlling the specificity of the reaction. In order to generate the required number of PCR product molecules. . To improve the PCR amplification of DNA fragments.

7-0. • Increase MgCl2 concentration up to 3-4.0) 2.dNTPs mix (25 mM each nucleotide) 4.7µL 2. but keep MgCl2 concentration at 1.Taq DNA polymerase (native enzyme) 6.0µL FINAL CONCENTRATION 1x 200 µM (each nucleotide) 0. QUESTIONS 1. • 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. but keep MgCl2 concentration at 1.10x PCR Buffer* 3.Troubleshooting discussion is based on the PCR protocol as described in the table below. COMPONENT 1. 15 mM MgCl2 (the final concentrations of these ingredients in the PCR mix are: 50 mM KCl.2x-2x.4µL 0. 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 1.52mM.primer mix (25 pmoles/µL each primer) 5.52mM • Increase MgCl2 concentration up to 3-4.5µL 0.8x.5 mM but keep dNTP concentration constant. 1. 100 mM Tris-HCl (pH 8.genomic DNA template (100 ng/µL) VOLUME 20.5 mM MgCl2). 10 mM Tris-HCl.2µL 1.3).autoclaved ultra-filtered water (pH 7. All reactions are run for 30 cycles. I get (many) shorter unspecific products. I get (many) longer unspecific products.2µL 0. 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 0.4 µM (each primer) 1 Unit/25 µL 100 ng/25 µL * The 10x PCR buffer contains: 500 mM KCl.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 .

Can I run a multiplex PCR with them?. • 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. use BSA (0. How many loci can I amplify in multiplex PCR at the same time? • Difficult to say. • If some of the loci are weak or not amplified.8 µg/µL final concentration). If you need to keep the size of the product constant. yes. The balance between these amounts is more important than the absolute values used !!. I have a number of primer pairs I would like to use together. check all your PCR ingredients. • Check primer sequences for primer-primer interactions 9. You can also try 5% (v/v.3. If you do get products (including unspecific ones) reaction conditions as described above. • Combine some/all of the above 4. How? • Very likely. Taq. read below !! 7. Best. What can I do to improve PCR amplification? • An easy solution is to increase the length of the primer with low Tm. One or a few loci in my multiplex reaction are very weak or invisible. The author has routinely amplified from 2 to 14 loci. Short PCR products in my multiplex reaction are weak. but now I can't get any product. • Make sure all PCR ingredients are taken in the reaction (buffer. 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. add a few bases at either the 3' or the 5' end of that primer. How can I improve their yield? . My PCR product is weak. If one of the primer pairs yields unspecific products. final concentration) DMSO or glycerol. 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. • Try amplify all loci seaprately using the same PCR program. • 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 you don't. 8. 6. Is there a way to increase the yield? • Gradually decrease the annealing temperature to the lowest possible. If size is not a concern. My two primers have very different melting temperatures (Tm) but I cannot change their locus. keep the cycling conditions constant and change other parameters as mentioned above (#1 and #2). add a few bases at the 3' end. template.1 to 0. Reaction was working before. etc) • Change the dNTP solution (very sensitive to cycles of thawing and freezing. especially in multiplex PCR) • If you just bought new primers. • Check primer sequences for mismatches and/or increase the primer length by 5 nucleotides • Combine some/all of the above 5. • Literature describes up to 25 loci or so.

use BSA (0.1 to 0.1 to 0. use BSA (0.8 µg/µL final concentration). . • Add adjuvants. using an annealing temperature lower than usual. Can I get rid of them somehow? • If long: increase buffer concentration to 1. Best. Best.• • • • • • • Increase KCl (buffer) concentration to 1. Add adjuvants. final concentration) DMSO or glycerol • If nothing works: run PCR reactions for each (multiplexed) locus individually. Best. but keep MgCl2 concentration at 1. Longer PCR products in my multiplex reaction are weak. • 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. final concentration) DMSO or glycerol Combine some/all of the above 10. You can also try 5% (v/v. use BSA (0. How can I improve their yield? • Decrease KCl (buffer) concentration to 0. use BSA (0. This may indicate which primer pair yields the unspecific products in the multiplex reaction.2-2x.8 µg/µL final concentration). Unspecific products appear in my multiplex reaction. final concentration) DMSO or glycerol • Combine some/all of the above 11.52mM • Gradually increase the annealing temperature • Decrease amount of template • Decrease amount of primer • Decrease amount of enzyme • Increase MgCl2 concentration up to 3-4.7-0.5-2mM • If short: decrease buffer concentration to 0.1 to 0. Compare the unspecific products for each locus tested with the unspecific products seen when running the multiplex PCR. but keep MgCl2 concentration at 1. but keep MgCl2 concentration at 1. All products in my multiplex reaction are weak. but keep MgCl2 concentration at 1. 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.1 to 0.8 µg/µL final concentration).8x.7-0.9x.5 mM but keep dNTP concentration constant.52mM • Increase MgCl2 concentration up to 3-4. You can also try 5% (v/v. You can also try 5% (v/v.5-2mM • Increase MgCl2 concentration up to 3-4. but keep MgCl2 concentration at 1.2x-2x.5 mM but keep dNTP concentration constant • Add adjuvants.5 mM but keep dNTP concentration constant. You can also try 5% (v/v. final concentration) DMSO or glycerol • Combine some/all of the above 12. Best.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.8 µg/µL final concentration).

• • 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) .