PCR Reagents

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Case Study

• Hot Start PCR with Hot Start Taq Antibody (anti-Taq DNA polymerase antibody)

  Hot Start PCR is a widely used technique that inhibits Taq DNA polymerase activity during PCR reaction preparation until a heat activation happens. Antibody-medicated, chemical modification and aptamer-medicated methods are all feasible to limit the Taq DNA polymerase activity. GenScript offers proprietary Hot Start Taq Antibody (anti-Taq DNA polymerase antibody) for antibody-medicated Hot Start PCR. Hot Start PCR improves specificity of target gene fragment amplification, as well as increases the yield of targeted fragment.

  Taq DNA polymerase (Cat. No. E00007) and Hot Start Taq Antibody (Cat. No. A01849) mixtures were used in PCR reactions. The amplified target DNA fragment is 500 bp from HPRT gene. The PCR products were analyzed by agarose gel (see figure 1). The non-specific PCR products were observed in the lanes without Hot Start Taq Antibody added, and fewer non-specific PCR products were amplified when higher units of Hot Start Taq Antibody used. Hence, Hot Start Taq Antibody-medicated Hot Start PCR can notably improve the specificity of target gene amplification and increase the yield of PCR products.

  

  Figure 1: PCR products analysis by agarose gel. The Hot Start Taq Antibody (Cat. No. A01849) used in lane 1 and 2 is 2 U, in Lane 3 and 4 is 1 U, in Lane 5 and 6 is 0.5 U, and in lane 7 and 8 is none.

FAQ

• What is the recommended amount of Taq DNA Polymerase or Green Taq DNA Polymerase to use?

  A: .5 U or more.

• How is Taq different from Green Taq?

  A: The Green Taq contains Taq polymerase with a special stabilizer that allows it to be stable at room temperature for 2 weeks.

• Can Taq DNA Polymerase or Green taq be used to amplify GC-rich amplicons?

  A: No.

• Can Taq DNA Polymerase be used in other buffers?

  A: The Taq DNA polymerase is 100% active in the buffer supplied by GenScript, but also works in competitors' buffers. For your convenience, we sell 10 x Taq buffer Cat. No. B0005.

• Which buffer should I use if I want to control the level of magnesium in the reaction? Or does the presence of Mg²⁺ inhibit PCR?

  A: We suggest using Mg-free Taq DNA Polymerase (Cat. No. E00008).

• How should I set up an amplification reaction using Taq DNA Polymerase?

  A: Here are the guidelines for 50 μl PCR reaction:

  • 0.5 μl Taq DNA Polymerase
  • 1 μl 10 mM dNTP
  • 1 μl each primer
  • 2 μl genomic template (up to 100 ng/μl)
  • 5 μl 10 X Taq Buffer (Standard Taq containing Mg²⁺)
  • 39.5 μl sterile or filtered H₂O
  • Denature at 94°C
  • Extend 1 minute/kb
• What is the recommended concentration of Taq Polymerase to use for a routine PCR reaction?

  A:

  • For up to 3 kb, we recommend 1.25 units per 50 µl reaction.
  • For specialized applications, including 3–8 kb amplicons, 2.5 units per 50 µl reaction is recommended.
• What is the upper limit for length of the product when using Taq DNA Polymerase from GenScript?

  A: Up to λdna 8 kb.

• What type of DNA ends result from a primer extension reaction or a PCR using Taq DNA Polymerase?

  A: The Poly A tail contains sticky ends, and can only do TA cloning.

• Why is there no product when visualized on an agarose gel?

  A: There may be a problem in the PCR program or system, or a problem during the electrophoresis.

• If the product sequence doesn't completely match the expected sequence. How can this result be improved?

  A: We suggest to use a high-fidelity DNA Polymerase which can offer the highest fidelity of any commercial thermostable polymerase.

• When should Taq DNA Polymerase be used in a primer extension reaction or for PCR?

  A: The Taq can be used when the PCR reaction started or after degeneration.

• Will the 5'→3' flap endonuclease activity of Taq DNA Polymerase degrade primers?

  A: No. The exonuclease will only degrade double stranded DNA that it encounters while extending a DNA fragment. It will degrade a secondary primer if bound to the same strand (e.g. a mutagenesis primer).

• Where can I find help troubleshooting my PCR?

  A: Please send all your issue via [email protected], or load the protocol of the Taq DNA polymerase direcly.

Citations

• Citations
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  • Y Acanda., et al. EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine. Plant Cell Rep.2013 Dec.
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  • Zhou P., et al. IGF-I Signaling Is Essential for FSH Stimulation of AKT and Steroidogenic Genes in Granulosa Cells. Mol Endocrinol.2013 Mar;27(3):511 - 523.
  • Wang X., et al. Silencing of the Host Factor eIF (iso) 4E Gene Confers Plum Pox Virus Resistance in Plum. PLoS One.2013 Jan;8(1):e50627.
  • Chiorazzi M., et al. Related F-box proteins control cell death in Caenorhabditis elegans and human lymphoma. Proc Natl Acad Sci U S A.2013 Mar;110(10):3943 - 3948.
  • Doyle VP., et al. Habitat and Host Indicate Lineage Identity in Colletotrichum gloeosporioides sl from Wild and Agricultural Landscapes in North America. PLoS One.2013 May;6(5):e62394.
  • Jun TH., et al. Genetic mapping of three quantitative trait loci for soybean aphid resistance in PI 567324. Heredity (Edinb).2013 Jul;111(1):16-22.
  • Carol D. von Dohlen., et al. Diversity of proteobacterial endosymbionts in hemlock woolly adelgid (Adelges tsugae)(Hemiptera: Adelgidae) from its native and introduced range. Environ Microbiol.2013 Jul;15(7):2043-62.
  • Gandhirajan RK., et al. Blockade of NOX2 and STIM1 signaling limits lipopolysaccharide-induced vascular inflammation. J Clin Invest.2013 Feb;123(2):887-902.
  • Zhou JB., et al. Identification of a novel gene fusion RNF213â€'SLC26A11 in chronic myeloid leukemia by RNA-Seq. Mol Med Rep.2013 Feb;7(2):591-7.
  • Jessica Hoppstdter., et al.Glucocorticoid-induced leucine zipper is downregulated in human alveolar macrophages upon Toll-like receptor activation. Eur J Immunol.2012 May;42(5):1282-93.
  • Sheng H., et al. Mapping QTL for resistance to eyespot of wheat in Aegilops longissima. Theor Appl Genet.2012 Jul;125(2):355-366.

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