John M. S. Bartlett.pdf - Bio-Nica.info

296 Rithidech and Dunn
11. Ethidium bromide (0.5 µg/mL).
12. DNA size standards.
3. Methods
Basically, two basic principles are involved in this protocol, that is, 1) the repeat
sequences are amplified by PCR by using primers specific for the flanking genes and
2) the amplicons are then sized on nondenaturing polyacrylamide gels. The protocol
outlined below has been used successfully for genotyping 50 mouse microsatellite
markers, included in our study on leukemogenesis and hepatoma, of BALB/cJ X
CBA/CaJ hybrid mice. The sizes of these markers are 84 to 270 bp with allelic size
differences of 8 to 30 bp. The following steps are required.
3.1. Step 1
Set up PCR Master Mix. An example given in below is for a single PCR amplification
of three microsatellite markers. To make a total volume of 15 µL Master Mix, combine
the appropriate set of components in order listed in below in a 0.5-mL thin-walled
PCR tube.
Volume (µL)
Final Concentration
Sterile Milli Q Water 1.19
10× PCR buffer 1.5 1X
dATP at 10 mM 0.3 200 µM
dCTP at 10 mM 0.3 200 µM
dGTP at 10 mM 0.3 200 µM
dTTP at 10 mM 0.3 200 µM
Microsatellite marker 1:
Forward Primer (6.6 µM) 1.14 0.5 µM
Reverse Primer (6.6 µM) 1.14 0.5 UM
Microsatellite marker 2:
Forward Primer (6.6 µM) 1.14 0.5 µM
Reverse Primer (6.6 µM) 1.14 0.5 µM
Microsatellite marker 3:
Forward Primer (6.6 µM) 1.14 0.5 µM
Reverse Primer (6.6 µM) 1.14 0.5 µM
MgCl 2 (25 mM) 1.2 2.0 mM
template DNA (5 µg/mL) 3.0 1.0 µg/mL
Taq polymerase (5Units/µL) 0.072 2.5 Units/100 µL
NOTES:
1. All chemicals, except primers, are purchased from Perkin-Elmer, Norwalk, CT.
2. All primers are purchased from Research Genetics, Inc. Huntsville, AL.
Add template DNA after a drop of mineral oil (Sigma, St. Louis, MO) has been placed
into the tube.
Add after “Hot Start” in Step 2.
3.2. Step 2
Hot-start at 96°C for 3 min. Then, add 0.072 µL of Taq polymerase (5 U/µL).