Pcr how much taq

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Our mission is to develop high-quality innovative tools and services to accelerate discovery. We use cookies to improve your browsing experience and provide meaningful content. Read our cookie policy. Download: PCR enzyme brochure. New products. Contact Sales. Optimizing your PCR PCR can sometimes require optimization of reaction conditions in order to obtain a successful result. When optimizing PCR conditions, which conditions are particularly important? Initial denaturation step Preheating is sometimes required to denature complex templates e.

PrimeSTAR enzymes do not require preheating for enzyme activation. Denaturing conditions Denaturing conditions should be selected by considering the thermal cycler model that will be used.

Annealing conditions The annealing step should be adjusted for each primer set; the annealing temperature depends directly on the T m of primers.

For Taq enzymes, the recommended annealing time is 30 sec. Therefore, it is important to use a short annealing time of 5—15 sec. Excessively long annealing times may lead to mispriming-induced nonspecific amplification. When amplifying short sequences smaller than 1 kb, a three-step PCR protocol is recommended.

High-copy targets, such as housekeeping genes, require only 10 ng of template. Template amounts for higher-complexity templates range between 10 ng and ng. The amount of cDNA template depends on the copy number of the target. A general recommendation is to start with standard concentrations and adjust as necessary. Higher primer concentrations often contribute to mispriming and nonspecific amplification.

On the other hand, low primer concentrations can result in low or no amplification of the desired target Figure 3. Figure 3. Note the accumulation of nonspecific products and primer dimers with high primer concentrations. These four nucleotides are typically added to the PCR reaction in equimolar amounts for optimal base incorporation. However, in certain situations such as random mutagenesis by PCR, unbalanced dNTP concentrations are intentionally supplied to promote a higher degree of misincorporation by a non-proofreading DNA polymerase.

For efficient incorporation by DNA polymerase, free dNTPs should be present in the reaction at a concentration of no less than 0. Figure 4. The final concentration of MgCl 2 in each reaction was 4 mM. In some applications, the dNTPs may include special nucleotides. Figure 5. Figure 6. However, some polymerases such as Pfu DNA polymerase prefer MgSO 4 , since sulfate helps ensure more robust and reproducible performance under certain circumstances. Figure 7. PCR amplification with various concentrations of MgCl 2.

The top bands represent the desired 2. PCR is carried out in a buffer that provides a suitable chemical environment for activity of DNA polymerase. The buffer pH is usually between 8. Note that DNA polymerases often come with PCR buffers that have been optimized for robust enzyme activity; therefore, it is recommended to use the provided buffer to achieve optimal PCR results.

Figure 8. Effects of buffer ions on DNA duplex formation. Figure 9. PCR results from varying concentrations of MgCl 2 in two different buffer types, illustrating importance of buffer choice for PCR specificity.

In certain scenarios, chemical additives or co-solvents may be included in the buffer to improve amplification specificity by reducing mispriming and to enhance amplification efficiency by removing secondary structures Table 2.

These reagents are commonly used with difficult samples such as GC-rich templates. Also, they can interfere with certain downstream applications— for example, nonionic detergents in microarray experiments. Hence, it is important to be aware of buffer compositions for successful PCR and downstream usage. 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. However, their use will also neutralize the inhibitory affects of SDS, an occasional contaminant of DNA extraction protocols. Hot start PCR is a versatile modification in which the initial denaturation time is increased dramatically Table 4. This modification can be incorporated with or without other modifications to cycling conditions.

Moreover, it is often used in conjunction with additives for temperamental amplicon formation. In fact, hot start PCR is increasingly included as a regular aspect of general cycling conditions. Hot start has been demonstrated to increase amplicon yield, while increasing the specificity and fidelity of the reaction.

The rationale behind hot start PCR is to eliminate primer-dimer and non-specific priming that may result as a consequence of setting up the reaction below the T m.

In general, the DNA polymerase is withheld from the reaction during the initial, elongated, denaturing time. Although other components of the reaction are sometimes omitted instead of the DNA polymerase, here we will focus on the DNA polymerase. There are several methods which allow the DNA polymerase to remain inactive or physically separated until the initial denaturation period has completed, including the use of a solid wax barrier, anti-DNA polymerase antibodies, and accessory proteins.