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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
4.
Amplification Reactions
Materials to Be Supplied by the User
(Solution compositions are provided in Section 7.A.)
•
nuclease-free light mineral oil
•
Perkin-Elmer Model 480 or GeneAmp
®
PCR System 9600 or 9700 thermal cycler
•
Amplification tubes compatible with thermal cycler. For the Perkin Elmer Model 480 thermal cycler, use 0.5ml
thin-walled GeneAmp
®
reaction tubes. For the GeneAmp
®
PCR System 9600 and 9700 thermal cyclers, use
0.2ml thin-walled MicroAmp
®
reaction tubes or a MicroAmp
®
optical 96-well reaction plate.
•
4% NuSieve
®
3:1 Plus TBE Buffer agarose precast Reliant
®
minigels (Cambrex
•
Cat.# 54927, 54928 or 54929), Latitude
®
precast minigels agarose (Cambrex
•
Cat.# 57222 or 57232), or NuSieve
®
3:1 agarose (Cambrex Cat.# 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 bromide
solutions.
4.A. Reaction Setup for Large Sample Numbers
This protocol is designed to analyze multiple samples at one time. If you are analyzing only 1 or 2 samples, see
Section 7.C.
In this procedure:
•
Aliquots of the sample DNAs are placed in the reaction tube.
•
Taq
DNA polymerase is added to each of the Multiplex Master Mixes.
•
The Multiplex Master Mixes containing
Taq
DNA polymerase are added to the reaction tubes. Each sample is
analyzed with all five Multiplex Master Mixes.
Protocol
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 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 a negative no-DNA
control for each Multiplex Master Mix.
!
