What is the difference between native and recombinant Taq?

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Answer

Native Taq DNA polymerase is purified from the thermophile Thermus aquaticus. Recombinant Taq is produced from the polymerase gene and is purified from E. coli. This procedure separates a HMW form of the enzyme (94,000 kDa) from a LMW form of the enzyme (87,000 kDa)---recombinant Taq DNA Polymerase comprises only 94,000 kDa form. This 94,000 kDa form is much more stable and exhibits a small amount of 5' to 3' exonuclease activity. The recombinant Taq DNA Polymerase expressed in E. coli is almost identical to native Thermus aquaticus with respect to activity, specificity, thermostability and performance in PCR. The major difference between the two forms is in their thermostability at 95 degrees C.

Answer Id: E4060

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b0e477bf95926f2fc02b0ab41bc47621_FAQ

How does does Platinum® Taq DNA Polymerase differ from regular Taq Polymerase?

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Answer

Platinum® Taq DNA Polymerase is designed for hot-start PCR. Because it is bound by a monoclonal antibody, Platinum® Taq DNA Polymerase is inactive when the temperature is below 94 degrees C. The high-temperature step at the beginning of the PCR cycle (typically 94 degrees C) denatures and dissociates the antibody from the polymerase, restoring it to full activity.

Mispriming typically occurs at the start of PCR. Using a hot-start polymerase such as Platinum® Taq DNA Polymerase dramatically reduces mispriming, as it has little to no activity until after the denaturation step. Therefore, hot-start PCR--mediated by Platinum® Taq DNA Polymerase--has a higher specificity than PCR with standard Taq DNA Polymerase.

Answer Id: E2974

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aa50d32d1893d194901c7828ed10f1dc_FAQ

What is the source and purity of your Muristerone A product (H100-01)?

Product FAQ

Answer

Muristerone A is purified from plant sources.

Purity (HPLC): 94.0%
Formula: C27H44O8
Molecular Weight: 496.6
Solubility: Soluble in methanol, ethanol, acetic acid and DMSO. Sparingly soluble in chloroform. Insoluble in water.
Recommended Storage: Keep cool and dry at -20 C.

Answer Id: E3516

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d1cb3472bf551c3fc39a26de42e4ee86_FAQ

What is the molecular weight and size of Taq Polymerase?

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Answer

Taq DNA Polymerase is a 832 amino acid, single subunit enzyme with a MW of 94,000. (Reference: Lawyer (1989);JBC 264, 6427.)

Answer Id: E4059

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af9a78431c5b1a9baa20c62ca83e6201_FAQ

What is the difference between Platinum® technology and AccuPrime™ technology?

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Answer

With Platinum® technology, anti-DNA polymerase antibodies bind to the enzyme until the denaturing step at 94 degrees C, when the antibodies degrade. The polymerase is now active and primer extension can occur. AccuPrime™ Taq combines Platinum® Taq (Taq + Platinum® antibodies) with proprietary thermostable AccuPrime™ accessory proteins. The 10X reaction buffer contains the accessory proteins which enhance specific primer-template hybridization during each cycle of PCR.

Answer Id: E7266

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bd08d795d08f482150b59b7ec27d03d7_FAQ

What are the main steps in PCR?

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Answer

The main steps are: denaturation, annealing, and extension. The template is typically heated to a high temperature (around 94-95 degrees C) allowing for the double-stranded DNA to denature into single strands. Next, the temperature is lowered to 50-65 degrees C, allowing primers to anneal to their complementary base-pair regions. The temperature is then increased to 72 degrees C, allowing for the polymerase to bind and synthesize a new strand of DNA.

Answer Id: E7269

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3358bfd4156788bcf689aa8838c5d372_FAQ

The primers I am using worked for PCR initially, but over time, have stopped working. What happened?

Product FAQ

Answer

Primers should be aliquoted for single use before PCR set-up. Heat just the aliquoted primers to 94 degrees for 1 min. Quick chill the primer on ice before adding to the PCR reaction. Some primers may anneal to themselves or curl up on themselves.

Answer Id: E7302

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03de3389616245442315d9da95048050_FAQ

Do you have any references on Dynabeads® FlowComp™ Mouse CD4 kit and Dynabeads® FlowComp™ Mouse CD8 kit?

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Answer

Brody JD et al. (2009) Immunotransplantation preferentially expands T-effector cells over T regulatory cells and cures large lymphoma tumors. Blood 113:85-94.

Feng X et al. (2010) Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development. Blood 115:510-518.

Yi JS et al. (2010) IL-21 deficiency influences CD8 T cell quality and recall responses following an acute viral infection. J Immunol 185:4835-4845.

Answer Id: E6139

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233660772cce9dca261dce959350590f_FAQ

What is the function of the antibody in Platinum® Taq? How does this contribute to hot-start PCR?

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Answer

Platinum® Taq DNA Polymerase is recombinant Taq DNA polymerase complexed with a proprietary antibody that inhibits polymerase activity. The activity of Platinum® Taq DNA polymerase is blocked at ambient temperatures but is regained after the denaturation step in PCR cycling at 94 degrees C. This property makes Platinum® Taq DNA polymerase a hot-start reagent. Hot starts are typically used in PCR to increase sensitivity, specificity, and yield--and hot-start enzymes allow users to set up reactions at room temperature. By using a hot-start polymerase, it improved PCR results can be obtained with less optimization and handling of reaction components.

Answer Id: E4052

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ccccf44b4758aa4c967a5567f9e8fe43_FAQ

What is the pH of the NuPAGE® LDS Sample Buffer (4X) at 70°C?

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Answer

The temperature coefficient (ΔpKa/degree C) for Tris is -0.031/degree C. Therefore, if the pH of the NuPAGE® LDS Sample Buffer is 8.5 at 25°C, then its pH at 70°C is 8.5 + (-0.031 X 45) = 7.1. Similarly, our Tris Glycine Sample Buffer at 85°C would have a pH of 4.94, at 100°C its pH would be 4.5.

Answer Id: E3874

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f5b794b250d4ea8cfb3b41d0c8e298e4_FAQ

How should the probe be applied to the slides?

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Answer

Depending on the size of the tissue section, a starting volume of 15 µL of the probe can be applied either to the coverslip or straight on to the tissue section with a pipette. The sides between the slide and the coverslip are then sealed with rubber cement. A convenient alternative is the use of CISH UnderCover™ Slips (Cat. No. 00-8403 or 00-8404).
If the side of the slides are not to be sealed, it is important to perform subsequent incubations at 37 degrees C in a humidified chamber. Denature the probe on a thermal plate (at 94-95 degrees C).

Answer Id: E5003

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e6e8bdd6b6b17bbb47919a3095944e9d_FAQ

How does the protocol used in the TOPO® XL kit improve the efficiency of cloning long PCR products?

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Answer

The protocol includes a novel 15-minute gel purification step to improve the efficiency of cloning long PCR products. Gel purification eliminates smaller PCR products which preferentially ligate into the vector. In addition, the TOPO® XL PCR cloning kit protocol eliminates the use of ethidium bromide and UV light which can nick long PCR products and reduce cloning efficiency. The kit uses the non-toxic crystal violet reagent to allow visualization of DNA under ambient light. In side-by-side cloning experiments, long PCR products that were purified after visualization with crystal violet yielded 94% recombinant colonies versus only 60% with ethidium bromide staining and UV light visualization.

Answer Id: E6752

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a8688302e454ba1db20d14068a92f38f_FAQ

Why is coupling efficiency important?

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Answer

Coupling efficiency is important as the effects are cumulative during DNA synthesis. The numbers below shows the effect of a 1% difference in coupling efficiency and how this influences the amount of full-length product available following synthesis of different length oligos. Even with a relatively short oligo of 20 bases, a 1% difference in coupling efficiency can mean 15% more of the DNA present following synthesis is full-length product.

Number of bases added, 99% coupling full-length, Failures, 98% coupling full-length, Failures:
- 1, 99, 1, 98, 2
- 2, 98.01, 1.99, 96.04, 2.96
- 3,97.03, 2.97, 94.12, 5.88
- 10, 90.44, 9.56, 81.71, 18.29
- 20, 81.79, 18.21, 66.76, 33.24
- 30, 73.79, 26.03, 54.55, 63.58
- 50, 60.5, 39.5, 36.42, 63.58
- 95, 38.49, 61.51, 14.67, 85.33

Answer Id: E7278

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30ecfec787d38116a9b3ccf51cf8126c_FAQ

Manual: GeneAmp PCR System 2700 Quick Reference Card (English )

Manual / Product Insert

Version: 20 Jun 2007
7928930515844730f8e63b0634226df6_Manuals