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Introduction 1-11

be purchased individually (P/N N808-0096) or as part of the SYBR

Green Core Reagents Kit (P/N 4304886).

Fluorescent

Contaminants

Since fluorescent contaminants can interfere with SYBR Green I

assays and give false-positive results, it may be necessary to include a

No Amplification Control tube that contains sample, but no enzyme. If

the absolute fluorescence of the No Amplification Control is greater

than that of the No Template Control after PCR, fluorescent

contaminants may be present in the sample or in the heat block of the

thermal cycler.

Prevention of PCR

Product Carryover

Use primers that contain dA nucleotides near the 3´ ends so that any

primer-dimer generated is efficiently degraded by AmpErase UNG at

least as well as any dU-containing PCR products. The further a dA

nucleotide is from the 3´ end, the more likely that partially degraded

primer-dimer molecules may serve as templates for a subsequent PCR

amplification.

Production of primer dimer could lower the amplification yield of the

desired target region. If primers cannot be selected with dA nucleotides

near the ends, the use of primers with 3´ terminal dU-nucleotides

should be considered. Single-stranded DNA with terminal dU

nucleotides are not substrates for AmpErase UNG (Delort et al., 1985)

and thus the primers will not be degraded. Biotin-dUMP derivatives are

not substrates for AmpErase UNG.

The concentration of AmpErase UNG and the time of the incubation

step necessary to prevent amplification of contaminating dU-containing

PCR product depends on the PCR conditions necessary to amplify your

particular DNA sequence and the level of contamination expected. In

most cases, using AmpErase UNG at 1 U/l00

µL reaction and

incubation at 50 °C for two minutes is sufficient.

Do not attempt to use AmpErase UNG in subsequent amplification of

dU-containing PCR template, such as in nested-PCR protocols. The

UNG will degrade the dU-containing PCR product, preventing further

amplification.

General PCR

Practices

When preparing samples for PCR amplification:

Wear a clean lab coat (not previously worn while handling amplified

PCR products or used during sample preparation) and clean

gloves.

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