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
Multiplex
Master Mix
# of Sample
DNAs
Positive Control
(Male Genomic
DNA)
Negative Control
(No DNA)
Total Reaction
Tubes
A
1
1
B
1
1
C
1
1
D
1
1
E
1
1
3.
Set up and label the required number of reaction tubes as determined above. Use thin-walled amplification tubes
or an optical plate. Place on ice.
4.
In a separate tube, dilute each sample DNA to 10ng/µl using the supplied Nuclease-Free Water. Mix well by
vortexing for 5–10 seconds. Add 5µl of diluted DNA to the appropriately labeled reaction tubes (above) on ice.
Dilute the Male Genomic DNA to 10ng/µl by adding 6µl of Male Genomic DNA to 24µl of Nuclease-Free Water.
Mix well by vortexing for 5–10 seconds. Add 5µl to appropriately labeled reaction tubes (above) on ice.
For the negative control (no DNA), add 5µl of Nuclease-Free Water to the appropriately labeled reaction tubes
(above) on ice.
5.
Prepare five Multiplex Master Mix and
Taq
DNA polymerase mixtures on ice, one for each Multiplex Master Mix.
Vortex the Multiplex Master Mixes before using them.
Component
Volume per
Reaction
Volume per
10 Reactions
Multiplex Master Mix
20µl
200µl
Taq
DNA Polymerase (5u/µl)
0.2µl
2µl
Final Volume
20.2µl
202µl
6.
Vortex to mix.
7.
Add 20µl of the Multiplex Master Mix and
Taq
DNA polymerase mixture to the appropriate reaction tubes,
which contain the sample DNA or controls, on ice.
8.
Gently vortex to mix.
9.
Centrifuge briefly to bring the contents to the bottom of the tubes. Place on ice until ready for thermal cycling.
10. 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.
