14
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TM248 · Revised 12/18
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5.
Troubleshooting (continued)
Symptoms
Causes and Comments
Dropout of bands in
Suboptimal conditions. The system is tested using the Perkin-
amplification of sample DNA (continued)
Elmer GeneAmp
®
PCR System 9600, 9700 and the Model 480
thermal cyclers. If you are using a different thermal cycler,
you will need to optimize amplification conditions.
EDTA in sample. EDTA at a concentration of ≥0.4mM can
adversely affect amplification.
Incorrect protocol. The protocol must be followed precisely.
Wrong reaction volume. Standard reactions are 25µl.
Reactions of 12.5µl are prone to dropout of multiple bands.
Bands in negative control reactions
Cross-contamination. Pre- and post-amplification areas
should be isolated, preferably in separate rooms. Use aerosol-
resistant tips and dedicated pipettes for assembling reactions.
Workstations and pipettes can be cleaned with a mild bleach
solution before and after use.
6. References
1.
Skaletsky, H.
et al.
(2003) The male-specific region of the human Y chromosome is a mosaic of discrete sequence
classes.
Nature
423
, 825–37.
* Especially note Figure 2, available online as data supplementary to the article.
2.
Vollrath, D.
et al
. (1992) The human Y chromosome: A 43-interval map based on naturally occurring deletions.
Science
258
, 52–9.
3.
Foote, S.
et al.
(1992) The human Y chromosome: Overlapping DNA clones spanning the euchromatic region.
Science
258
, 60–6.
4.
Affara, N.
et al.
(1996) Report of the second international workshop on Y chromosome mapping 1995.
Cytogenet. Cell. Genet
.
73
, 33–76.
5.
Kent-First, M.G.
et al.
(1996) Gene sequence and evolutionary conservation of human SMCY.
Nat. Genet.
14
,
128–9.
6.
Vogt, P.H.
et al.
(1997) Report of the Third International Workshop on Y-chromosome mapping 1997.
Cytogenet. Cell Genet.
79
, 1–20.
7.
Kostiner, D.R., Turek, P.K. and Reijo, R.A. (1998) Male infertility: Analysis of the markers and genes on the
human Y chromosome.
Hum. Reprod.
13
, 3032–8.
8.
Lahn, B.T. and Page, D.C. (1997) Functional coherence of the human Y chromosome.
Science
278
, 675–80.
9.
Reijo, R.
et al.
(1996) Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y
chromosome.
Lancet
347
, 1290–3.
