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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 

3

www.promega.com

  

TM248 · Revised 12/18

2. 

Product Components and Storage Conditions

P R O D U C T  

S I Z E   C AT. #

Y Chromosome Deletion Detection System, Version 2.0 

25 reactions 

MD1531

For Research Use Only. Not for use in diagnostic procedures. The Y Chromosome Deletion Detection System, 
Version 2.0, includes:

• 

500µl 

Multiplex A Master Mix

• 

500µl 

Multiplex B Master Mix

• 

500µl 

Multiplex C Master Mix

• 

500µl 

Multiplex D Master Mix

• 

500µl 

Multiplex E Master Mix

• 200u 

GoTaq

®

 DNA Polymerase

• 

1ml 

Nuclease-Free Water

• 

90µg 

50bp DNA Step Ladder (340ng/µl)

• 

2.5µg 

Male Genomic DNA (50ng/µl)

• 

1ml 

Blue/Orange 6X Loading Dye

Storage Conditions: 

Store all components at –20°C. Avoid multiple freeze-thaw cycles. The Nuclease-Free Water 

may be stored at room temperature (22–25°C).

3. 

System Requirements

3.A. Template

DNA concentration, purity and size are important considerations to ensure success with the Y Chromosome Deletion 
Detection System, Version 2.0. DNA should be free of contaminating protein and salts. The DNA should not be 
sheared. Poor-quality DNA may result in increased background or amplification failure. Adding too much or too little 
DNA to the reaction can cause amplification failure. Failure can be manifested in several ways; sometimes complete 
lack of amplification of all bands is observed with all master mixes, and in other cases dropout of all or portions of the 
alleles in individual master mixes will be observed. Several commercially available DNA purification systems, including 
the Wizard

®

 Genomic DNA Purification Kit (Cat.# A1120, A1125, A1620) and the MagneSil

®

 KF, Genomic System 

(Cat.# MD1460), produce DNA of sufficient quality for the Y Chromosome Deletion Detection System.

Quantitation of DNA

We recommend quantitating the DNA prior to use in the Y Chromosome Deletion Detection System; either too much or 
too little DNA can cause the reactions to fail. Absorbance readings at 260nm can be used to estimate DNA 
concentration where 1Au = 50µg of double-stranded DNA/ml. Fifty nanograms of DNA should be used in each 
amplification reaction.

Summary of Contents for MD1531

Page 1: ...Revised 12 18 TM248 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...

Page 2: ...oduct Components and Storage Conditions 3 3 System Requirements 3 3 A Template 3 3 B Thermal Cyclers 4 3 C Contamination Control 4 3 D Control Reactions 5 4 Amplification Reactions 6 4 A Reaction Setu...

Page 3: ...x E contains a control primer pair that amplifies a unique region in both male and female DNA ZFX ZFY These control primer pairs are internal controls for the amplification reaction and the integrity...

Page 4: ...o ensure success with the Y Chromosome Deletion Detection System Version 2 0 DNA should be free of contaminating protein and salts The DNA should not be sheared Poor quality DNA may result in increase...

Page 5: ...sity of the bands indicate different DNA concentrations that result from variation in the A260 A280 ratio Figure 2 Gel image of 100ng based on A260 genomic DNA of varying quality The end lanes contain...

Page 6: ...taminated with DNA Figure 1 lanes 2 4 6 8 and 10 Figure 3 Schematic representation of the Y Chromosome Deletion Detection System assay Sample DNA Positive Control Male Genomic DNA Negative Control No...

Page 7: ...at 50090 or 50091 1X TBE buffer ethidium bromide aerosol resistant pipette tips UV transilluminator Caution Ethidium bromide is a suspected carcinogen Always wear gloves when working with ethidium bro...

Page 8: ...vortexing for 5 10 seconds Add 5 l to appropriately labeled reaction tubes above on ice For the negative control no DNA add 5 l of Nuclease Free Water to the appropriately labeled reaction tubes above...

Page 9: ...o preheat the instrument to 94 C before placing tubes into the machine Program for the Perkin Elmer Model 480 Thermal Cycler Preheat the thermal cycler to 94 C before placing tubes inside Cycling Prof...

Page 10: ...e recommend using a 4 NuSieve 3 1 Plus TBE buffer precast gel Alternatively cast a 4 NuSieve 3 1 agarose gel in 1X TBE buffer containing 0 5 g ml ethidium bromide For instructions for the preparation...

Page 11: ...ed from the ZFY ZFX genes The absence of these products is indicative of a problem with that particular PCR amplification If the control bands are present with the Male Genomic DNA control but not wit...

Page 12: ...n products on the agarose gel If your negative controls show no detectable PCR products these nonspecific bands should have no effect on analysis Figure 4 Y chromosome deletion analysis The amplificat...

Page 13: ...kin 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 opt...

Page 14: ...le 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...

Page 15: ...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 not...

Page 16: ...P1193 Mineral Oil 12ml DY1151 DNA Purification Systems Product Size Cat Wizard Genomic DNA Purification Kit 100 isolations 300 l A1120 500 isolations 300 l A1125 100 isolations 10ml A1620 MagneSil KF...

Page 17: ...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...

Page 18: ...h the beaker or mark the level of the liquid in the beaker 5 Mix to wet agarose 6 Soak the agarose for 15 minutes at room temperature 7 Heat the beaker in a microwave until the solution boils 8 Boil t...

Page 19: ...imental sample Multiplex A Master Mix Samples STS Locus Size of Product bp Map Position SY254 DAZ 380 18 SY157 DYS240 290 20 SY81 DYS271 209 2 SY130 DYS221 173 11 SY182 KAL Y 125 5 SMCX 83 Control Mul...

Page 20: ...ega com TM248 Revised 12 18 Multiplex D Master Mix Samples STS Locus Size of Product bp Map Position SY133 DYS223 177 12 SY152 DYS236 125 15 SY124 DYS215 109 8 SMCX 83 Control Multiplex E Master Mix S...

Page 21: ...eports of a positive SY133 signal in some samples with confirmed AZFb deletions suggesting possible genetic variants for this locus 4320MA09_3A SRY DYS271 DYS148 DYS273 KALY DYS212 SMCY DYS215 DYS218...

Page 22: ...13 2014 2017 2018 Promega Corporation All Rights Reserved GoTaq MagneSil and Wizard are registered trademarks of Promega Corporation GeneAmp is a registered trademark of Roche Molecular Systems Inc La...

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