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
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www.promega.com
TM248 · Revised 12/18
Symptoms
Causes and Comments
Low yield or no
The thermal cycler was not preheated prior to placing tubes
amplification product (continued)
inside. Preheat the instrument to 94°C.
Multiple nonspecific
Thermal cycler programmed incorrectly. Verify that the times
amplification products
and temperatures are correct.
Reactions were not set up on ice. Make sure to set up reactions
on ice.
Too much genomic DNA. We recommend using 50ng of DNA
per reaction.
Too much
Taq
DNA polymerase. Do not use more than 1 unit
of
Taq
DNA polymerase per reaction.
Annealing temperature too low. Check the accuracy of the
thermal cycler. Increase the annealing temperature in
0.5–1°C increments.
Missing bands in control reaction
Annealing temperature too high. Check the accuracy of the
thermal cycler. Decrease the annealing temperature in 0.5–1°C
increments.
Internal control band is missing in
Sample DNA degraded or of poor quality. Repeat reactions.
sample DNA but present in control
You may need to re-isolate sample DNA. We recommend the
Wizard
®
Genomic DNA Purification Kit or MagneSil
®
KF,
Genomic System.
Dropout of bands in
Too little DNA used. DNA should be quantitated prior to use
amplification of sample DNA
in the Y Chromosome Deletion Detection System. Use 50ng
for each amplification reaction or 250ng total for each sample.
DNA concentration can be overestimated by spectrophotometry
if the A
260
/A
280
ratio is low. Verify DNA concentration 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 DNA purity. High-quality DNA typically has an
A
260
/A
280
ratio of ≥1.8. However, A
260
/A
280
ratio may not
accurately predict purity. Impurities in the sample may cause
amplification failure. To test for impurities, add some of your
sample DNA to the positive control. Subsequent inhibition of
this “spiked” positive control reaction indicates that
impurities are in the sample DNA.
