How long pcr extension

Have you ever wondered who came up with that thermocycler protocol we all use? While the denaturation and annealing times remain rather constant across the board, the extension time can vary significantly based on the size of amplicon you are generating. The general rule of thumb is to allow for a 60 second extension time per 1 kb of your amplicon. For shorter amplicons, reduce the extension time.

For a bp amplicon, an extension time of 15 to 20 seconds is sufficient. Too long of an extension time can introduce unwanted products! However, there are other enzymes that work more or less efficiently for various templates. When you purchase your polymerase enzyme, an optimized buffer will likely accompany it. However, it is important to understand the components of that buffer and their contributions to the PCR efficiency. Magnesium concentration can be manipulated to alter the efficiency and fidelity of the PCR.

For Taq DNA polymerase, the optimal magnesium concentration is 1. Because other buffer and reaction components may chelate the magnesium, it may be necessary to increase the magnesium concentration. Thus, 1 ng contains 2. The working stocks were then used to generate the Master Mix solutions outlined in Table 7.

Experiments varied cycling conditions as described below. No MgCl 2 was present in the original PCR buffer and had to be supplemented at the concentrations indicated with a range tested from 0. The recommended concentration provided by the manufacturer was 1. Perhaps surprisingly, the necessary concentration needed for product formation in this experiment exceeded this amount. A different DNA template was used for the experiment presented in Figure 3b.

As shown in Figure 3b , amplification of the desired PCR product requires at least 2. Notice that in the experiments presented in Figures 3A and 3B , a discrete band was obtained using the cycling conditions thought to be optimal based on primer annealing temperatures.

For the third experiment presented in Figure 3c , three changes were made to the cycling conditions used to amplify the yeast GAL3 gene. Second, the extension time was extended to 1 minute and 30 seconds. Third, the number of cycles was increased from 30 to 35 times. The purpose was to demonstrate the effects of sub-optimal amplification conditions i.

As shown in Figure 3c , what was a discrete band in Figure 3a , becomes a smear of non-specific products under these sub-optimal cycling conditions. These results also demonstrate that when both the cycling conditions are correctly designed and the reagents are at optimal concentrations, the PCR experiment produces a discreet amplicon corresponding to the expected size.

The results show the importance of performing PCR experiments at a sufficiently high stringency e. Moreover, the experiments indicate that changing one parameter can influence another parameter, thus affecting the reaction outcome. The Master Mix depicted in the above table is calculated for 11 reactions plus 2 extra reactions to accommodate pipette transfer loss ensuring there is enough to aliquot to each reaction tube.

Table 7. Figure 1. Note that primers do not always anneal at the extreme ends and may form smaller loop structures. Once the primers anneal to each other they will elongate to the primer ends. Figure 2. Ice bucket with reagents, pipettes, and racks required for a PCR. P pipette, 2. P pipette, 3. P pipette, 4. P pipette, 5. Figure 3. Lanes 9 - 11 are indicative of excessively stringent conditions with no product formed. Figure 4. Sterile tubes used for PCR.

Figure 5. Thermal cycler. Closed thermal cycler left image. Right image contains 0. PCR has become an indispensible tool in the biological science arsenal. PCR has altered the course of science allowing biologists to yield power over genomes, and make hybrid genes with novel functions, allowing specific and accurate clinical testing, gaining insights into genomes and diversity, as well as simply cloning genes for further biochemical analysis. PCR application is limited only by the imagination of the scientist that wields its power.

There are many books and papers that describe new specialized uses of PCR, and many more will be developed over the next generation of biological science.

However, regardless of the anticipated approaches, the fundamental framework has remained the same. PCR, in all its grandeur, is an in vitro application to generate large quantities of a specific segment of DNA. Designing a PCR experiment requires thought and patience. The results shown in Figure 3 exemplify one of the major challenges when designing an optimization strategy for PCR. That is, as one parameter of PCR is changed, it may impact another. An attempt to resolve the smear might involve setting up PCR conditions with reactions containing 2.

However, as seen in Figure 3a, this would not yield any product. Consequently, it is advisable to titrate reagents, rather than adding one concentration to a single reaction, when troubleshooting spurious results. If all else fails, redesign the primers and try, try again. I would also like to thanks Giancarlo Costaguta and Gregory S. I would also like to thank Bhairav Shah for taking pictures of the lab equipment and reagents used to make figures 2 - 4. National Center for Biotechnology Information , U.

J Vis Exp. Published online May Todd C. Lorenz 1. Author information Copyright and License information Disclaimer. Correspondence to: Todd C. Lorenz at ude. This article has been cited by other articles in PMC.

Abstract In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. Keywords: Basic Protocols, Issue 63, PCR, optimization, primer design, melting temperature, T m , troubleshooting, additives, enhancers, template DNA quantification, thermal cycler, molecular biology, genetics.

Download video file. Protocol 1. Designing Primers Designing appropriate primers is essential to the successful outcome of a PCR experiment. Below is a list of characteristics that should be considered when designing primers. Primer length should be nucleotide residues bases. Notes: There are many computer programs designed to aid in designing primer pairs.

Organize laboratory equipment on the workbench. Setting up a Reaction Mixture Start by making a table of reagents that will be added to the reaction mixture see Table 1. Next, label PCR tube s with the ethanol-resistant marker. Notes: When setting up multiple PCR experiments, it is advantageous to assemble a mixture of reagents common to all reactions i.

I use a hot start method for all of my L-PCR. Each fraction is 1X for buffer concentration. Alternatively, after denaturing, an 80 degree step can be used for adding the polymerase fraction P. Table 1 qPCR assay features.

Data analysis Data were analysed using instrument default settings and quantification cycles Cqs were calculated automatically. Table 2 Quantification cycles recorded at each of the different qPCR reaction conditions. Conflict of interest There is no conflict of interest.

Acknowledgement All experiments were carried out and analysed by the author. Notes Handled by Jim Huggett. References 1. Saiki R. Kopp M. Chemical amplification: continuous-flow PCR on a chip. Noncontact infrared-mediated thermocycling for effective polymerase chain reaction amplification of DNA in nanoliter volumes.

Zhang C. Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends. Nucleic Acids Res. Wheeler E. Under-three minute PCR: probing the limits of fast amplification. Fuchiwaki Y. Ultra-rapid flow-through polymerase chain reaction microfluidics using vapor pressure. Montgomery J. The influence of nucleotide sequence and temperature on the activity of thermostable DNA polymerases.