Organize laboratory equipment on the workbench. When setting up several PCR experiments that all use the same reagents, they can be scaled appropriately and combined together in a master mixture Master Mix. This step can be done in a sterile 1. To analyze the amplicons resulting from a PCR experiment, reagents and equipment for agarose gel electrophoresis is required. To approximate the size of a PCR product, an appropriate, commercially available molecular weight size standard is needed.
Setting up a Reaction Mixture Start by making a table of reagents that will be added to the reaction mixture see Table 1. Reaction volumes will vary depending on the concentrations of the stock reagents. Add only if it is not present in the 10X buffer or as needed for PCR optimization.
For example, to obtain the 4. Add 10 4 to 10 7 molecules or about 1 to ng DNA template. However, it should be noted that water is added first but requires initially making a table of reagents and determining the volumes of all other reagents added to the reaction. Allowing PCR reagents to be added into cold 0.
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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. The following website offers a calculator for determining the number of copies of a template DNA http: The total number of copies of double stranded DNA may be calculated using the following equation: 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.
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While most modern PCR machines use 0. See your thermal cyclers manual to determine the appropriate size tube. 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.
However, nearest-neighbor thermodynamic models are preferred over the more conventional calculation: 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. For calculating the T m values by nearest-neighbor thermodynamic models, one of the following calculators is recommended: 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 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. 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. Manipulating PCR Reagents 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.
Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies
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.
This convenient, spiral-bound, laboratory manual saves readers valuable time by providing easily accessible information on key topics and protocols. Succinctly describes the most commonly applied techniques and contains useful tips on stopping points, troubleshooting, and safety. Essential Techniques Series PCR is one of the most important techniques in modern molecular biology and many variants, based on DNA amplification, have been developed.
This book provides detailed practical guidance on both the established and new applications of PCR for a wide range of disciplines, for example gene analysis, DNA sequencing, forensics and diagnostics. It will be essential reading for all those using PCR, whatever their field of interest. The Essential Techniques Series books are designed to provide you with immediate access to the protocols you require every day.
These handy pocket-sized manuals are easy to carry around, and conveniently spiral bound making them ideal for lab bench work.
Written by experienced laboratory researchers, each book in the Essential Techniques Series gives up-to-date, tried and tested practical information for the life scientist. For each key technique these books: Would you like to tell us about a lower price? If you are a seller for this product, would you like to suggest updates through seller support? Learn more about Amazon Prime. Read more Read less. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature Tm of the primers.
To check whether the PCR successfully generated the anticipated DNA target region also sometimes referred to as the amplimer or amplicon , agarose gel electrophoresis may be employed for size separation of the PCR products. As with other chemical reactions, the reaction rate and efficiency of PCR are affected by limiting factors. Thus, the entire PCR process can further be divided into three stages based on reaction progress:. In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products.
Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as formamide , in buffer systems may increase the specificity and yield of PCR. Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid , phage , or cosmid depending on size or the genetic material of another organism.
Bacterial colonies such as E. Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing Fig. This technique may also be used to determine evolutionary relationships among organisms when certain molecular clocks are used i. This is often critical for forensic analysis , when only a trace amount of DNA is available as evidence.
These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth , and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian tsar and the body of English king Richard III. RT-qPCR allows the quantification and detection of a specific DNA sequence in real time since it measures concentration while the synthesis process is taking place. There are two methods for simultaneous detection and quantification.
The first method consists of using fluorescent dyes that are retained nonspecifically in between the double strands. The second method involves probes that code for specific sequences and are fluorescently labeled. Detection of DNA using these methods can only be seen after the hybridization of probes with its complementary DNA takes place. An interesting technique combination is real-time PCR and reverse transcription. This sophisticated technique allows for the quantification of a small quantity of RNA.
This technique lowers the possibility of error at the end point of PCR,  increasing chances for detection of genes associated with genetic diseases such as cancer. Prospective parents can be tested for being genetic carriers , or their children might be tested for actually being affected by a disease. PCR analysis is also essential to preimplantation genetic diagnosis , where individual cells of a developing embryo are tested for mutations.
PCR allows for rapid and highly specific diagnosis of infectious diseases, including those caused by bacteria or viruses. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes. Characterization and detection of infectious disease organisms have been revolutionized by PCR in the following ways:. PCR has a number of advantages. It is fairly simple to understand and to use, and produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis.
Therefore, it has its uses to analyze alterations of gene expression levels in tumors, microbes, or other disease states. PCR is a very powerful and practical research tool. The sequencing of unknown etiologies of many diseases are being figured out by the PCR. The technique can help identify the sequence of previously unknown viruses related to those already known and thus give us a better understanding of the disease itself.
If the procedure can be further simplified and sensitive non radiometric detection systems can be developed, the PCR will assume a prominent place in the clinical laboratory for years to come.
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One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification. Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR fragments that are generated. Another limitation of PCR is that even the smallest amount of contaminating DNA can be amplified, resulting in misleading or ambiguous results. To minimize the chance of contamination, investigators should reserve separate rooms for reagent preparation, the PCR, and analysis of product.
Reagents should be dispensed into single-use aliquots. Pipetters with disposable plungers and extra-long pipette tips should be routinely used. Gobind Khorana first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. There, he was responsible for synthesizing short chains of DNA.