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Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
TM248 · Revised 12/18
www.promega.com
3.A. Template (continued)
DNA Quality
High-quality DNA typically has an A
260
/A
280
ratio of ≥1.8. The presence of impurities in the DNA sample can cause
amplification failure. We have observed that DNA concentration can be overestimated by spectrophotometry if the
A
260
/A
280
ratio is low. When low amounts of DNA are added to the Y Chromosome Deletion Detection System, band
dropout in one or more of the master mixes can be observed. The amount of DNA in a sample can be verified by
ethidium bromide staining of DNA following electrophoresis in a 1% agarose gel. One hundred nanograms of genomic
DNA should be visible on an ethidium bromide-stained gel visualized by UV transillumination. The Male Genomic
DNA standard supplied in the Y Chromosome Deletion Detection System can be used as an electrophoresis standard.
Figure 2 is an example of an agarose gel containing ethidium bromide-stained genomic DNA samples of 100ng of DNA
as determined by A
260
. Differences in the intensity of the bands indicate different DNA concentrations that result from
variation in the A
260
/A
280
ratio.
Figure 2. Gel image of 100ng (based on A
260
) genomic DNA of varying quality.
The end lanes contain 100ng
of Male Genomic DNA supplied with the system.
3.B. Thermal Cyclers
The Y Chromosome Deletion Detection System has been optimized for use with either the Perkin-Elmer GeneAmp
®
PCR System 9600 or 9700 or the Perkin-Elmer Model 480 thermal cycler. Use of other thermal cyclers may require
optimization of cycling parameters. We recommend validating with actual samples in addition to the positive control,
as the preparation of the sample DNA can affect the cycling conditions. The Perkin-Elmer Model 480 thermal cycler
program is a good starting point for thermal cyclers without programmed ramps, and the Perkin-Elmer GeneAmp
®
PCR System 9700 cycling program is a good starting point for thermal cyclers with programmed ramps.
3.C. Contamination Control
Preventing DNA contamination of reaction components is essential. Always use aerosol-resistant pipette tips and clean
gloves, and avoid carryover contamination of stock solutions. Reagents that are used for amplification reactions should
be maintained and used separately from completed amplification reactions, preferably in separate pre-amplification
and post-amplification locations.
