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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
5. Troubleshooting
For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information
available at:
www.promega.com;
E-mail:
Symptoms
Causes and Comments
Low yield or no
Thermal cycler programmed incorrectly. Verify that the times
amplification product
and temperatures are correct.
Thermal cycler not properly calibrated. Perform a set of
control reactions to determine if certain positions in the
thermal cycler yield little or no product.
Top of thermal cycler open. The top must be closed for correct
heating and cooling.
Missing reaction component. Check the reaction components,
and repeat the reaction.
Degraded reagent. Store Multiplex Master Mixes at –20°C.
Keep on ice once thawed. Avoid multiple freeze-thaw cycles.
Incorrect tubes. Use only thin-walled microcentrifuge tubes
for amplification. For the Perkin-Elmer Model 480 thermal
cycler, use 0.5ml thin-walled GeneAmp
®
reaction tubes, and
for the GeneAmp
®
PCR System 9600 or 9700 thermal cycler,
use 0.2ml thin-walled MicroAmp
®
reaction tubes or
MicroAmp
®
optical 96-well reaction plate.
Inhibitors in the DNA sample. DNA should be free of
contaminating proteins and salts. Repurify or ethanol
precipitate the DNA sample to remove inhibitors.
Too much or too little DNA used. DNA should be quantitated
prior to use in the Y Chromosome Deletion Detection System.
Use 50ng of high-molecular-weight (nonsheared) DNA for
each amplification reaction or 250ng total for each sample.
Inaccurate quantitation of DNA. DNA concentration can be
overestimated by spectrophotometry if the A
260
/A
280
ratio is
low. Verify DNA concentration and quality by ethidium
bromide staining after electrophoresis on a low percent
(1–1.2%) agarose gel. We recommend comparing sample
DNA with genomic DNA of known concentration such as the
Male Genomic DNA supplied with the system. One hundred
nanograms of the Male Genomic DNA is clearly visible on
ethidium bromide-stained gels.
Poor mixing of components. Concentration gradients can
form in Multiplex Master Mixes stored at –20°C. Mix
thoroughly by vortexing for 10–15 seconds before use.
