Paul.xu: Created page with "1) 0.01% Phenol red (R. J. Klebe, S. A. Rodriguez, M. L. VerBeek and T. A.
Giambernardi, 1999. Simple method for "hot-start" RT-PCR. BioTechn.
27(6):1108-1110.)
2)..."

2015-06-19T20:24:43Z

Created page with "1) 0.01% Phenol red (R. J. Klebe, S. A. Rodriguez, M. L. VerBeek and T. A. Giambernardi, 1999. Simple method for "hot-start" RT-PCR. BioTechn. 27(6):1108-1110.) 2)..."

New page

1) 0.01% Phenol red (R. J. Klebe, S. A. Rodriguez, M. L. VerBeek and T. A. Giambernardi, 1999. Simple method for "hot-start" RT-PCR. BioTechn. 27(6):1108-1110.) 2) Carl Wittwer's laboratory uses 0.1 mM Cresol Red for the same purpose (The Rapid Cyclist, Spring, 1994, page 9). Neither dye will act as a loading buffer, but serves as an indication that the Taq has been added and mixed properly. A scan of various websites looking at their RedTaq storage buffers provides the clue: 3) One company claims the dye is chemically attached; I suspect that only means the dye binds to proteins. "Taq-red DNA Polymerase is Taq DNA polymerase with chemically attached chromogenic molecule. The enzyme is stained in red. The stain facilitate the visual control of mixing the enzyme with other components. The stain doesn't influence the enzyme activity and other general characteristics. Storage buffer 10 mM K-phosphate, pH7.4, 0.1 mM EDTA, 50% glycerol, 0.1% Triton X-100, 0.1% Tween 20." 4) Sigma's "REDTaq DNA Polymerase is a unique blend of Sigma's quality Taq DNA Polymerase combined with an inert red dye. This dye enables quick visual confirmation of enzyme addition and reaction mixing. An aliquot of the samples (5-10 µL) can then be loaded directly onto an agarose gel for electrophoresis following PCR. The red dye migrates slightly faster than bromophenol blue at about the same rate as a 125 base pair fragment in a 1% agarose gel. Since no additional loading buffers are added to the reaction following PCR, reamplification is possible. The red dye has no effect on automated or manual sequencing, restriction digestions or other downstream applications. However, if removing the dye is desired, this can easily be accomplished using any standard purification method." One clue: The REDTaq concentration is only 1 unit/µL, rather than the standard 5 units/uL used by other suppliers. Addition of a larger volume is required, so perhaps the extra 50% glycerol is acting as the agent that keeps the sample in the well? "Note: REDTaq 10X PCR Buffer has been formulated to be compatible with REDTaq. If other buffers are to be used, they must be formulated to account for 0.4 mM magnesium being added to the PCR from the enzyme. In this case the final enzyme concentration in the PCR is assumed to be 0.05 units per microliter (2.5 units per 50 microliter reaction). Other enzyme concentrations may require further magnesium concentration optimization. REDTaq DNA Polymerase, Product Code D 5684 1 unit/µl in 20 mM Tris-HCl, pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Igepal CA-630, inert dye, 50% glycerol." "Add the following reagents to a thin-walled 200 µl or 500 µl PCR tube in the order listed below. Amount Component 5 µL 10X REDTaq PCR Buffer (1x) 1 µL Deoxynucleotide Mix (final 200 µM (each dNTP) - µL Primer (0.1-0.5 µM) -µL Primer (0.1-0.5 µM) 2.5 µl REDTaq DNA Polymerase (final 0.05 unit/µL) - µL Template DNA (typically 10 ng) q.s. Water 50 µL Total reaction." So, 2.5 uL of REDTaq in a 50 uL reaction = 1.25 uL glycerol/50 uL reaction, for a final 2.5% concentration. The typical range of glycerol concentrations used for 1x loading buffers is 3-10%.

Sigma "Note: A minimum of 1.5 units of REDTaq DNA polymerase must be added per 50 µL reaction for unencumbered gel loading." 1.5 units = 0.75 µL glycerol/50 µL reaction, for a final 1.5% concentration. This reduced glycerol concentration is probably trickier to load on a gel. (Note: I originally posted this information at the NWFSC Forum.) 

[[Category:Protocols]]

Red Taq - Revision history

Paul.xu: Created page with "1) 0.01% Phenol red (R. J. Klebe, S. A. Rodriguez, M. L. VerBeek and T. A.Giambernardi, 1999. Simple method for "hot-start" RT-PCR. BioTechn.27(6):1108-1110.)2)..."

Paul.xu: Created page with "1) 0.01% Phenol red (R. J. Klebe, S. A. Rodriguez, M. L. VerBeek and T. A.
Giambernardi, 1999. Simple method for "hot-start" RT-PCR. BioTechn.
27(6):1108-1110.)
2)..."