Promega MD1531 Technical Manual Download Page 1

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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.

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...