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Summary of Contents for Promega MD1531
- Page 1 T E C H N I C A L M A N U A L Y Chromosome Deletion Detection System, Version 2.0 Instructions for Use of Product MD1531 For Research Use Only. Not for use in diagnostic procedures. Revised 12/18 TM248...
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Page 2: Table Of Contents
7.D. Preparation of Agarose Gels ...................... 17 7.E. Worksheets ..........................18 8. Summary of Changes ......................... 21 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 www.promega.com TM248 · Revised 12/18... -
Page 3: Detection System, Version
Step Ladder (Cat.# G4521). The reactions were performed on a Perkin-Elmer Model 480 thermal cycler. The gel is a 4% NuSieve 3:1 Plus TBE Buffer Reliant gel. ® ® 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... -
Page 4: Product Components And Storage Conditions
1Au = 50µg of double-stranded DNA/ml. Fifty nanograms of DNA should be used in each amplification reaction. 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 www.promega.com... -
Page 5: Thermal Cyclers
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... -
Page 6: Control Reactions
Agarose gel electrophoresis Figure 3. Schematic representation of the Y Chromosome Deletion Detection System assay. 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 www.promega.com TM248 · Revised 12/18... -
Page 7: Amplification Reactions
Multiplex Master Mix. Include a positive Male Genomic DNA control and a negative no-DNA control for each Multiplex Master Mix. 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... - Page 8 If using a thermal cycler that requires mineral oil, tilt the tubes and add one drop of oil to the side of the tube, letting the oil run down the side of the tube. 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 www.promega.com...
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Page 9: 9600 Or 9700 Thermal Cyclers
4°C soak After completion of thermal cycling protocol, store samples at –20°C. 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... -
Page 10: Agarose Gel Electrophoresis
Photograph the gel using a UV transilluminator (320nm). 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 www.promega.com... -
Page 11: Data Analysis-Controls
Multiplex B, and SY84 and SY86 of the DYS273 and DYS148 loci, which are represented as sequential amplification products in Multiplex E. 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... - Page 12 Multiplex A–D. Multiplex E shows no deletions.) The marker (M) lanes contain the 50bp DNA Step Ladder (Cat.# G4521). 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 www.promega.com...
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Page 13: Troubleshooting
Multiplex Master Mixes stored at –20°C. Mix thoroughly by vortexing for 10–15 seconds before use. 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... - Page 14 “spiked” positive control reaction indicates that impurities are in the sample DNA. 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 www.promega.com TM248 · Revised 12/18...
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Page 15: References
Reijo, R. et al. (1996) Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet 347, 1290–3. 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... -
Page 16: Appendix
• Each sample DNA is analyzed with all five of the Multiplex Master Mixes. 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 www.promega.com TM248 · Revised 12/18... - Page 17 Add 5µl genomic DNA or control mixture prepared in Step 5 to the appropriate reaction tubes containing the Multiplex Master Mixes on ice. Vortex to mix. 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...
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Page 18: Preparation Of Agarose Gels
Run gel at 5V/cm until the bromophenol blue dye front migrates to the bottom of the gel. 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 www.promega.com... -
Page 19: Worksheets
Product (bp) Position SY128 DYS219 SY121 DYS212 SY145 DYF51S1 SY255 SMCX Control 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... - Page 20 Product (bp) Position ZFX/ZFY Control SY14 SY134 DYS224 SY86 DYS148 SY84 DYS273 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 www.promega.com TM248 · Revised 12/18...
- Page 21 AZFb deletions, suggesting possible genetic variants for this locus. 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...
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Page 22: Summary Of Changes
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products. 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 www.promega.com...
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