Standard pcr program

Pipette the following PCR reagents in the following order into a 0. Since experiments should have at least a negative control, and possibly a positive control, it is beneficial to set up a Master Mix in a 1. In a separate 0. In addition, another reaction if reagents are available should contain a positive control using template DNA and or primers previously known to amplify under the same conditions as the experimental PCR tubes.

The micropipettor should be set to about half the reaction volume of the master mix when mixing, and care should be taken to avoid introducing bubbles. Put caps on the 0. Once the lid to the thermal cycler is firmly closed start the program see Table 2.

When the program has finished, the 0. PCR products can be detected by loading aliquots of each reaction into wells of an agarose gel then staining DNA that has migrated into the gel following electrophoresis with ethidium bromide.

If a PCR product is present, the ethidium bromide will intercalate between the bases of the DNA strands, allowing bands to be visualized with a UV illuminator. When setting up multiple PCR experiments, it is advantageous to assemble a mixture of reagents common to all reactions i. For instance, if there are 10 x 0. The reagents in the Master Mix are mixed thoroughly by gently pumping the plunger of a micropipettor up and down about 20 times as described above.

Each PCR tube receives an aliquot of the Master Mix to which the DNA template, any required primers, and experiment-specific reagents are then added see Tables 1 and 7. False positives may occur as a consequence of carry-over from another PCR reaction which would be visualized as multiple undesired products on an agarose gel after electrophoresis.

Therefore, it is prudent to use proper technique, include a negative control and positive control when possible. While ethidium bromide is the most common stain for nucleic acids there are several safer and less toxic alternatives. While most modern PCR machines use 0. See your thermal cyclers manual to determine the appropriate size tube.

Knowing the melting temperature T m of the primers is imperative for a successful PCR experiment. Although there are several T m calculators available, it is important to note that these calculations are an estimate of the actual T m due to lack of specific information about a particular reaction and assumptions made in the algorithms for the T m calculators themselves.

The former will give more accurate T m estimation because it takes into account the stacking energy of neighboring base pairs. The latter is used more frequently because the calculations are simple and can be done quickly by hand.

See Troubleshooting section for information about how various PCR conditions and additives affect melting temperature. PCR thermal cyclers rapidly heat and cool the reaction mixture, allowing for heat-induced denaturation of duplex DNA strand separation , annealing of primers to the plus and minus strands of the DNA template, and elongation of the PCR product.

Any longer than 3 minutes may inactivate the DNA polymerase, destroying its enzymatic activity. One method, known as hot-start PCR, drastically extends the initial denaturation time from 3 minutes up to 9 minutes.

This protocol modification avoids likely inactivation of the DNA polymerase enzyme. Refer to the Troubleshooting section of this protocol for more information about hot start PCR and other alternative methods. The next step is to set the thermal cycler to initiate the first of 25 to 35 rounds of a three-step temperature cycle Table 2. While increasing the number of cycles above 35 will result in a greater quantity of PCR products, too many rounds often results in the enrichment of undesirable secondary products.

The three temperature steps in a single cycle accomplish three tasks: the first step denatures the template and in later cycles, the amplicons as well , the second step allows optimal annealing of primers, and the third step permits the DNA polymerase to bind to the DNA template and synthesize the PCR product. The duration and temperature of each step within a cycle may be altered to optimize production of the desired amplicon. The time for the denaturation step is kept as short as possible.

Usually 10 to 60 seconds is sufficient for most DNA templates. The denaturation time and temperature may vary depending on the G-C content of the template DNA, as well as the ramp rate, which is the time it takes the thermal cycler to change from one temperature to the next. The temperature for this step is usually the same as that used for the initial denaturation phase step 1 above; e. The cycle concludes with an elongation step.

The temperature depends on the DNA polymerase selected for the experiment. Pfu DNA Polymerase is recommended for use in PCR and primer extension reactions that require high fidelity and requires 2 minutes for every 1 kb to be amplified. See manufacturer recommendations for exact elongation temperatures and elongation time indicated for each specific DNA polymerase. The final phase of thermal cycling incorporates an extended elongation period of 5 minutes or longer.

This last step allows synthesis of many uncompleted amplicons to finish and, in the case of Taq DNA polymerase, permits the addition of an adenine residue to the 3' ends of all PCR products. This modification is mediated by the terminal transferase activity of Taq DNA polymerase and is useful for subsequent molecular cloning procedures that require a 3'-overhang.

The stringency of a reaction may be modulated such that the specificity is adjusted by altering variables e. For example, if the reaction is not stringent enough, many spurious amplicons will be generated with variable lengths. If the reaction is too stringent, no product will be produced. Troubleshooting PCR reactions may be a frustrating endeavor at times. However, careful analysis and a good understanding of the reagents used in a PCR experiment can reduce the amount of time and trials needed to obtain the desired results.

However, before changing anything, be sure that an erroneous result was not due to human error. Start by confirming all reagents were added to a given reaction and that the reagents were not contaminated.

Are there non-specific products bands that migrate at a different size than the desired product? Was there a lack of any product? Also, it is wise to analyze the G-C content of the desired amplicon. First determine if any of the PCR reagents are catastrophic to your reaction. This can be achieved by preparing new reagents e. This process will determine which reagent was the culprit for the failed PCR experiment.

In the case of very old DNA, which often accumulates inhibitors, it has been demonstrated that addition of bovine serum albumin may help alleviate the problem. Primer dimers can form when primers preferentially self anneal or anneal to the other primer in the reaction. If this occurs, a small product of less than bp will appear on the agarose gel.

Start by altering the ratio of template to primer; if the primer concentration is in extreme excess over the template concentration, then the primers will be more likely to anneal to themselves or each other over the DNA template. Adding DMSO and or using a hot start thermal cycling method may resolve the problem. In the end it may be necessary to design new primers. Non-specific products are produced when PCR stringency is excessively low resulting in non-specific PCR bands with variable lengths.

This produces a ladder effect on an agarose gel. It then is advisable to choose PCR conditions that increase stringency. A smear of various sizes may also result from primers designed to highly repetitive sequences when amplifying genomic DNA. However, the same primers may amplify a target sequence on a plasmid without encountering the same problem. Lack of PCR products is likely due to reaction conditions that are too stringent. Primer dimers and hairpin loop structures that form with the primers or in the denatured template DNA may also prevent amplification of PCR products because these molecules may no longer base pair with the desired DNA counterpart.

If the G-C content has not been analyzed, it is time to do so. However, there are many additives that have been used to help alleviate the challenges.

Understanding the function of reagents used on conventional PCR is critical when first deciding how best to alter reaction conditions to obtain the desired product. However, the wrong concentration of such reagents may lead to spurious results, decreasing the stringency of the reaction. When troubleshooting PCR, only one reagent should be manipulated at a time. However, it may be prudent to titrate the manipulated reagent.

Changing the magnesium concentration is one of the easiest reagents to manipulate with perhaps the greatest impact on the stringency of PCR. The 10 X PCR buffer solutions may contain 15 mM MgCl 2 , which is enough for a typical PCR reaction, or it may be added separately at a concentration optimized for a particular reaction.

If the desired amplicon is below bp and long non-specific products are forming, specificity may be improved by titrating KCl, increasing the concentration in 10 mM increments up to mM. Thus, choosing an appropriate enzyme can be helpful for obtaining desired amplicon products.

The addition of a 3' adenine has become a useful strategy for cloning PCR products into TA vectors whit 3' thymine overhangs. However, if fidelity is more important an enzyme such as Pfu may be a better choice. Several manufactures have an array of specific DNA polymerases designed for specialized needs. Take a look at the reaction conditions and characteristics of the desired amplicon, and then match the PCR experiment with the appropriate DNA polymerase.

Most manufactures have tables that aid DNA polymerase selection by listing characteristics such as fidelity, yield, speed, optimal target lengths, and whether it is useful for G-C rich amplification or hot start PCR. Optimal target molecules are between 10 4 to 10 7 molecules and may be calculated as was described in the notes above.

Additive reagents may yield results when all else fails. Understanding the reagents and what they are used for is critical in determining which reagents may be most effective in the acquisition of the desired PCR product. Adding reagents to the reaction is complicated by the fact that manipulation of one reagent may impact the usable concentration of another reagent.

In addition to the reagents listed below, proprietary commercially available additives are available from many biotechnology companies. Formamide final reaction concentration of 1.

Formamide also has been shown to be an enhancer for G-C rich templates. As the amplicon or template DNA is denatured, it will often form secondary structures such as hairpin loops. Betaine final reaction concentration of 0. Non ionic detergents function to suppress secondary structure formation and help stabilize the DNA polymerase. Non ionic detergents such as Triton X, Tween 20, or NP may be used at reaction concentrations of 0.

The presence of non ionic detergents decreases PCR stringency, potentially leading to spurious product formation. Since we run many samples in our facility, this touchdown protocol allows us to use the same cycling conditions for every strain. If you do not want to use the touchdown protocol, or your thermocycler does not have a program for it, then you can choose to use a single annealing temperature.

You may need to test multiple annealing temperatures to find the one that works well for that particular primer set. This section can help you determine what other strains this protocol can be used for, and will give you some sense of how specific this protocol is. For example, if the protocol is for a generic Cre strain, you will see many Cre strains listed in this section. For this Il10 knockout, you can see that all 10 of the mouse strains listed express the same Il10 tm1Cgn allele, suggesting that this protocol is specific for that allele.

The melt curve analysis is a protocol that is based on a standard PCR assay and incorporates fluorescent dye and a melting protocol following traditional PCR cycling. Distinguishing the different PCR products is based on the relative fluorescence intensity with amplicon melting.

We use melt-curve analysis because it can save time and money when performing large-scale amounts of genotyping. While very similar to standard PCR protocols, there are a few additional considerations and method differences with melt curve analysis.

Need an introduction to melt curve analysis before moving forward? Review this introduction to advanced PCR methods for more background information. While the primers section for a melt curve analysis will be the same as for a standard PCR, the results section will tell you what results to expect when you run this protocol as a melt curve analysis. This information is provided in the form of melting temperatures, or Tm. Each peak corresponds to an amplified product with a characteristic melting temperature.

To run a melt curve analysis protocol, you will need to perform the PCR reaction in the presence of an intercalating dye, and use a thermocycler that can measure melting temperature. A: We use a Roche Light Cycler , however any thermocycler that can measure melting temperature should be sufficient. Q: I am seeing different melt curve temperatures Tm when I run this protocol in my facility.

What is going wrong? A: Melting temperatures can be variable, and may not be the same when run in a different facility with a different PCR setup. We provide the melting temperatures as a starting point, however best practice is to run samples with a known genotype wild type, heterozygous, homozygous and use those to determine the melting temperature with your PCR setup. Do not be surprised if your melting temperatures are several degrees different from what we have seen at JAX!

A: Even if a protocol is described to be a melt curve analysis, you may be able to run it on a gel instead. However, some primers sets work better as a melt curve analysis, and that may be noted on the protocol if so. We use cookies to personalize our website and to analyze web traffic to improve the user experience.

You may decline these cookies although certain areas of the site may not function without them. Please refer to our privacy policy for more information. What is Personalized Medicine? Genetics vs. Settings Allow essential cookies. This may be avoided by only adding enzyme after the initial denaturation, before the reaction cools to the chosen annealing temperature. Information is that "gems" may be substituted by Vaseline TM. Simply use a molar ratio of the two primers eg: primer 1 at 0.

This allows production of mainly ssDNA of the sense of the more abundant primer, which is useful for sequencing purposes or making ssDNA probes. Load 5 - 30ul of sample into wells of 0. It is best to include EthBr in the gel AND in the gel buffer , as post-electrophoresis staining can result in band smearing due to diffusion, and if there is no EthBr in the buffer the dye runs backwards out of the gel, and smaller bands are stripped of dye and are not visible.

Polyacrylamide gels can be silver stained by simple protocols for extreme sensitivity of detection. Gels can be blotted directly after soaking in 0. Bands blotted in this way are already covalently fixed onto nylon membranes, and simply need a rinse in 5xSSPE before prehybridisation. The example shown is of detection of Human papillomavirus type 16 HPV DNA amplified from cervical biopsy samples Williamson A-L, Rybicki EP Detection of genital human papillomaviruses by polymerase chain reaction amplification with degenerate nested primers.

J Med Virol Note how much more sensitive blotting is, and how much more specific the detection is. This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions. It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.