Editing Red Taq

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

Sigma "Note: A minimum of 1.5 units of REDTaq DNA polymerase must be added<br/>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.)<br/>


[[Category:Protocols]]