S7804 datasheet(12/32 Pages) ETC

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“Hot Start” PCR

The three sets of primers used in the CpG WIZ™ Methylation Assay are derived

from sequences closely related to each other, which introduces the possibility of

mispriming. In order to avoid this and other PCR-related artifacts, "hot start"

PCR is recommended. "Hot start" PCR permits the Taq polymerase to begin the

reaction only after the template and primers are in single-stranded form.

There are several modifications of the standard PCR protocol which allow a "hot

start" to occur. In one scenario, the PCR reaction mixture excluding the

polymerase can be overlaid with mineral oil prior to heating to 95°C. At the end

of the incubation, the enzyme is pipetted directly into the mixture under the

mineral oil. A second method involves the physical separation of the polymerase

and the rest of the PCR mix with a wax bead. The enzyme combines with the

rest of the reaction mixture only after the wax melts. In another variation, an

anti-Taq antibody inhibits the polymerase during reaction setup by forming a

complex with the Taq enzyme. Taq polymerase becomes active when the

complex is abolished due to antibody denaturation during the 95°C incubation.

Alternatively, a "hot start" enzyme can be used. Refer to the manufacturer's

instructions for enzyme activation protocol.

Note: Do not use a polymerase with 3'-5' exonuclease activity (i.e.

proofreading).Do not use a wax bead that contains Mg2+. Any extra Mg2+ added

to the reaction mixture produces suboptimal results.

Genomic Control DNAs

The methylated (red cap) and unmethylated (white cap) control DNAs must

undergo bisulfite modification prior to PCR amplification using the

CpGenome™ DNA Modification Kit (S7820). When used with their respective

U and M primer sets, a single PCR product of an expected size is obtained in

each case.

W control genomic DNA is used in the PCR and is NOT to be used in the

chemical modification step. When used with the W primer set, it is a positive

control for PCR amplification. Failure to generate a PCR product indicates a

general failure in the PCR reaction.

S7804 Datasheet(PDF) 12 Page - List of Unclassifed Manufacturers