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1-14 Introduction

When to Generate Dissociation Curves

The GeneAmp

5700 SDS, ABI PRISM

7900HT SDS,

ABI PRISM

7700 SDS, and ABI PRISM

7000 SDS can be set up to generate a

dissociation curve in either of these instances:

Immediately after the real-time PCR run

Independently of the real-time PCR run

Note In the presence of AmpErase UNG and dUTP, product degradation may

occur from a previously run PCR plate due to residual AmpErase UNG activity.

Note Refer to the appropriate SDS User’s Manual for further information on

generating a dissociation curve.

Note The 7700 instrument uses a separate Dissociation Curve Analysis

software that employs the multicomponent data exported from the SDS

software v 1.7a or later to display the dissociation curves for each sample.

Using Agarose

Gels to Check PCR

Product Purity

The absence of nonspecific amplification can be confirmed by

analyzing the PCR amplification products by agarose gel

electrophoresis.

To check PCR product purity with agarose gels:

Step Action

1 Load 12 to 15 µL of sample per well on an ethidium

bromide-stained 4% NuSieve 3:1 agarose gel.

CHEMICAL HAZARD. Ethidium bromide causes

eye, skin, and respiratory tract irritation and is a known mutagen

(i.e., it can change genetic material in a living cell and has the

potential to cause cancer). Always use adequate ventilation such

as that provided by a fume hood. Please read the MSDS, and

follow the handling instructions. Wear appropriate protective

eyewear, clothing, and gloves.

2 Run the gel:

For PCR fragments <100 bp, run the gel at 80 to 100 V for 45 to

60 min.

For PCR fragments 100 to 250 bp, run the gel at 100 to 115 V for

1 to 1.5 h.

3 Run samples 1/3 to 1/2 the length of the gel, without letting the dye

run off the bottom of the gel.

Use a UV lamp to check the migration of the samples.

WARNING

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