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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
TM248 · Revised 12/18
www.promega.com
7.C. Reaction Setup for Small Sample Numbers (continued)
An alternative protocol for large sample numbers is also provided (Section 4.A).
1.
Thaw the Multiplex Master Mixes. Vortex for 5–10 seconds, and store on ice. Thaw the Nuclease-Free Water and
Male Genomic DNA.
2.
Determine the number of reactions to perform for each Multiplex Master Mix. For each sample there will be five
reaction tubes, one for each Multiplex Master Mix. Include a positive Male Genomic DNA control and negative
no-DNA control for each Multiplex Master Mix.
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.
Label the required number of reaction tubes as determined above. 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 MicroAmp
®
optical 96-well reaction plate.
Place the reaction tubes on ice.
4.
Vortex the Multiplex Master Mixes for 5–10 seconds. Place 20µl of each Multiplex Master Mix in the
appropriately labeled reaction tubes on ice.
5.
For each DNA sample, prepare a genomic DNA mixture on ice following the table below.
Component
Single
Reactions
Duplicate
Reactions
Sample DNA
~250ng
~500ng
Taq
DNA polymerase (5u/µl)
1µl
2µl
Nuclease-Free Water to a final volume of
25µl
50µl
For the positive Male Genomic DNA control reactions, combine 5µl of Male Genomic DNA, 1µl of
Taq
DNA
polymerase (5u/µl) and 19µl of Nuclease-Free Water on ice. Vortex to mix.
For the negative no-DNA control, add 1µl
Taq
DNA polymerase (5u/µl) to 25µl of Nuclease-Free Water on ice.
Vortex gently to mix.
6.
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.