Magnesium is a required cofactor for Taq polymerase. Without adequate free magnesium, Taq is inactive. However, excess magnesium can reduce Taq fidelity. The amount of free magnesium in a given PCR reactions is highly variable.
Therefore, if you are having PCR problems you should empirically test a variety of magnesium concentrations to find the ideal concentration for your particular reaction. To do this, test magnesium concentrations from 1. Important Note: Multiple freeze-thaw cycles can cause magnesium chloride solutions to form concentration gradients. Therefore it is important to fully thaw and vortex your stock magnesium solution before each use.
Bovine serum albumin BSA is a common addition in several molecular biology applications, most notably restriction enzyme digestions and PCR. It is also reported to prevent reaction components from sticking to tube walls. Use up to 0. Therefore, like always, you need to empirically test these additives yourself.
I know… I know… not what you want to hear. Now stop whining and get back to the bench! Has this helped you? Then please share with your network. You may also note that Nonidet P is discontinued, but there are some replacements available.
BSA addition was repeated over 30 PCR cycles and the effect on yield was analyzed as described above. Control experiments where glycerol or distilled sterile water was added at every tenth cycle did not increase the PCR yield, indicating that the effects of BSA are not due to a change in the reaction volume or to BSA effectively acting as a molecular crowder, a property attributed to some of the effects of glycerol in PCR [ 13 ].
These results also suggested to us a way to improve the relatively low efficiency of using whole plasmid site-directed mutagenesis method described in the QuickChange Stratagene Mutagenesis Kit Stratagene to introduce mutation in some GC-rich DNA template here the tlp2 gene, a 2.
This type of result, characterized by a poor yield of mutagenesis, has previously been recognized as a common pitfall of this method [ 23 , 24 ]. In the overlap extension PCR, 2 sets of primer pairs are first used to amplify two DNA fragments to be fused that are produced with a short overlapping sequence with one another. A third amplification uses the amplification products of the first reactions as templates, along with the outermost reverse and forward primers to generate chimeric constructs [ 25 ].
Subsequent cloning and sequencing confirmed that the correct chimeric construct has been obtained. The gels were stained with ethidium bromide and photographed. On all panels, the following legend applies: red line, DMSO 7. The BSA PCR step protocol evaluated here demonstrate that high yield of traditionally difficult to amplify DNA fragments can be obtained, with a combination of primers of different sequence complementarity to the template they target and of different nucleotide length.
The reactions that were supplemented with BSA at intermediate steps followed the same cycling parameters as listed above, except that the cycle was manually paused after every tenth steps to allow BSA addition. In control reactions, these same cycling parameters were carried out with the addition of the same volume of sterile distilled water or glycerol added instead of BSA. Unless otherwise noted, the initial cycling step was repeated for fifteen cycles and the second cycling step was repeated for 20 cycles.
The reactions that were supplemented with BSA at intermediate steps followed the same cycling parameters as listed above, except that the cycle was manually paused after every five steps to allow BSA addition. The cycle was repeated 16times. Results were then viewed on an agarose gel and the bands corresponding to the target genes were cut out and extracted using the QIAquick Gel Extraction Kit Qiagen.
The extracted fragments were then added in a ratio to a reaction that was carried out using the same parameters that were used to amplify the individual fragments. The resulting PCR products loaded into a 1. The gel was then photographed, and densitometric quantification of amplification products was carried out using the NIS Elements Br 2. Each PCR was repeated at least 3 times and an average value and standard deviation was recorded from the image analysis.
PCR yield increase was calculated by subtracting the yield obtained in control PCR without any additives from the yield obtained in presence of the additive tested. The resulting number was then divided by the yield obtained in PCR without any additives to give the percentage of PCR yield increased due to a certain additive [ 10 ].
EF designed and carried out all experiments, participated in the interpretation of the results and wrote the first draft of the manuscript. GA participated in the design of the study, interpretation of the results and drafted the manuscript.
All authors read and approved the final manuscript. National Center for Biotechnology Information , U. BMC Res Notes. Published online May Eric M Farell 1 and Gladys Alexandre 1. Author information Article notes Copyright and License information Disclaimer. Eric M Farell: ude. Received Apr 10; Accepted May This article has been cited by other articles in PMC. Abstract Background While being a standard powerful molecular biology technique, applications of the PCR to the amplification of high GC-rich DNA samples still present challenges which include limited yield and poor specificity of the reaction.
Background Ever since the introduction of the Polymerase Chain Reaction [ 1 ], it has been one of the most often used tools in molecular biology, and has played a role in many of the major advances in Biology including cloning [ 2 ], mutagenesis [ 3 ], even with small amounts of DNA target [ 4 ]. 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. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. See Navigation. Table 1. General recommendations on designing PCR primers. Deoxynucleoside triphosphates dNTPs. Table 2. Common additives or co-solvents used as PCR enhancers, and their recommended final concentrations [6].
Dordrecht: Springer. Gene 93 1 — Anal Biochem 1 — J Biol Chem 30 : — Steitz TA A mechanism for all polymerases. Nature — In: Methods in molecular biology 2nd ed. Totowa: Humana Press. Learn more. Related products.
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