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-   -   Cresol red for PCR loading buffer... (http://www.molecularstation.com/forum/protocols-methods-forum/17598-cresol-red-pcr-loading-buffer.html)

Tim Fitzwater 08-05-2004 02:25 PM

Cresol red for PCR loading buffer...
 

There are published reports of two different red dyes used for this purpose.

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 無) 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/無, 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/痞 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 痞 or 500 痞 PCR tube in
the order listed below. Amount Component 5 無 10X REDTaq PCR Buffer (1x) 1
無 Deoxynucleotide Mix (final 200 然 (each dNTP) - 無 Primer (0.1-0.5 然) -
無 Primer (0.1-0.5 然) 2.5 痞 REDTaq DNA Polymerase (final 0.05 unit/無) -
無 Template DNA (typically 10 ng) q.s. Water 50 無 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 無 reaction for unencumbered gel loading." 1.5 units = 0.75 無
glycerol/50 無 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.)
Tim Fitzwater
SomaLogic

---

Kyle Legate 08-05-2004 05:47 PM

Cresol red for PCR loading buffer...
 
Tim Fitzwater wrote:
Why would someone care enough to go to such lengths? Normal convection
during the cycles is more than enough to mix the enzyme.




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