Manual: TFA Aminolink CE Phosphoramidite Improved Labeling Reagent: Model 38X and ABI 39X Nucleic Acid Synthesizers: User Bulletin 94 (English )

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What is the difference between native and recombinant Taq?

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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: 4060

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

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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: 2974

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6788076842014c83cedadbe6b0ba0314_FAQ

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

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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: 3516

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cf1cf43cba274ae7f413e864682b80f8_FAQ

What is the molecular weight and size of Taq Polymerase?

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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: 4059

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75806e8a1c04cad241934a374c1359c0_FAQ

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

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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: 6139

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618faa1728eb2ef6e3733645273ab145_FAQ

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

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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: 4052

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

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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: 3874

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2ed80f6311c1825feb854d78fa969d34_FAQ

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

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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: 6752

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751f6b6b02bf39c41025f3bcfd9948ad_FAQ

How should the probe be applied to the slides?

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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: 5003

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240ac9371ec2671ae99847c3ae2e6384_FAQ

Protocol: Applied Biosystems 2720 Thermal Cycler: Quick Reference Card: Rev B (English )

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

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Additional Document: VariantSEQr™ Resequencing Primers: List of Available Genes: Miscellaneous

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Manual: GeneAmp® PCR System 2700 For Amplification of Nucleic Acids: User Guide (English )

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Brochures & Specifications: Pesticides in the MRM catalogue of Cliquid™ Quant Software

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