Microsatellite Test PCR Reactions
Below are 6 microsatellite test reactions that I use when ever I try a new primer set or if my PCRs quit working. All conditions specified are for one reaction. Multiply by the number of reactions you plan on doing + a few extra to allow for pipetting error (multiply by 110 for a 96 well plate). Once you find the test reaction that works the best on the microsatellite loci that you are using, continue using that condition. I generally use 3µl of DNA for blood and tissue samples, 5µl of DNA for molted feather and predated eggshell samples, and 10µl of DNA for historic samples and samples that refuse to work at 3µl or 5µl.
Blood & Tissue (Road Kill, Plucked Feathers [with multiple tips], & Hatched Eggshell Membranes) Microsatellite & DNA Sexing PCR Reactions
TEST 2 TEST 3 TEST 4 TEST 5 TEST 6 1X Reaction 1X Reaction 1X Reaction 1X Reaction 1X Reaction 1X Reaction ddH2O 8.16µl 7.96µl 7.76µl 7.90µl 7.70µl 7.50µl MgCl2 0.8µl 1.0µl 1.2µl 0.8µl 1.0µl 1.2µl 10X Buffer 1.5µl 1.5µl 1.5µl 1.5µl 1.5µl 1.5µl dNTPs 1.2µl 1.2µl 1.2µl 1.2µl 1,2µl 1.2µl Primer 1 0.12µl 0.12µl 0.12µl 0.20µl 0.20µl 0.20µl Primer 2 0.12µl 0.12µl 0.12µl 0.20µl 0.20µl 0.20µl taq 0.1µl 0.1µl 0.1µl 0.20µl 0.20µl 0.20µl DNA 3.0µl 3.0µl 3.0µl 3.0µl 3.0µl 3.0µl Total Volume 15µl 15µl 15µl 15µl 15µl 15µl
TEST 1
Molted Feather & Predated Eggshell Membrane Microsatellite & DNA Sexing PCR Reactions
TEST 2 TEST 3 TEST 4 TEST 5 TEST 6 1X Reaction 1X Reaction 1X Reaction 1X Reaction 1X Reaction 1X Reaction ddH2O 6.16µl 5.96µl 5.76µl 5.90µl 5.70µl 5.50µl MgCl2 0.8µl 1.0µl 1.2µl 0.8µl 1.0µl 1.2µl 10X Buffer 1.5µl 1.5µl 1.5µl 1.5µl 1.5µl 1.5µl dNTPs 1.2µl 1.2µl 1.2µl 1.2µl 1,2µl 1.2µl Primer 1 0.12µl 0.12µl 0.12µl 0.20µl 0.20µl 0.20µl Primer 2 0.12µl 0.12µl 0.12µl 0.20µl 0.20µl 0.20µl taq 0.1µl 0.1µl 0.1µl 0.20µl 0.20µl 0.20µl DNA 5.0µl 5.0µl 5.0µl 5.0µl 5.0µl 5.0µl Total Volume 15µl 15µl 15µl 15µl 15µl 15µl
TEST 1
Historic Sample Microsatellite & DNA Sexing PCR Reactions
TEST 2 TEST 3 TEST 4 TEST 5 TEST 6 1X Reaction 1X Reaction 1X Reaction 1X Reaction 1X Reaction 1X Reaction ddH2O 1.16µl 0.96µl 0.76µl 0.90µl 0.70µl 0.50µl MgCl2 0.8µl 1.0µl 1.2µl 0.8µl 1.0µl 1.2µl 10X Buffer 1.5µl 1.5µl 1.5µl 1.5µl 1.5µl 1.5µl dNTPs 1.2µl 1.2µl 1.2µl 1.2µl 1,2µl 1.2µl Primer 1 0.12µl 0.12µl 0.12µl 0.20µl 0.20µl 0.20µl Primer 2 0.12µl 0.12µl 0.12µl 0.20µl 0.20µl 0.20µl taq 0.1µl 0.1µl 0.1µl 0.20µl 0.20µl 0.20µl DNA 10.0µl 10.0µl 10.0µl 10.0µl 10.0µl 10.0µl Total Volume 15µl 15µl 15µl 15µl 15µl 15µl
TEST 1
Microsatellite Test Duplex PCR Reactions
Below are 6 microsatellite test reactions that I use when I try to multiplex two different microsatellites. You can also do this for 3 microsatellites using all of the same volumes except adding 0.12 of each primer from primer set 3 (adjusting for a constant total volume is not important). All conditions specified are for one reaction. Multiply by the number of reactions you plan on doing + a few extra to allow for pipetting error. Once you find the test reaction that works the best, continue using that condition. I generally find that test #3 works best in most situations.
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|
TEST 1
|
TEST 2 |
TEST 3 |
TEST 4 |
TEST 5 |
TEST 6 |
|
|
1X Reaction |
1X Reaction |
1X Reaction |
1X Reaction |
1X Reaction |
1X Reaction |
|
ddH2O |
5.92µl |
5.72µl |
5.52µl |
5.64µl |
5.44µl |
5.20µl |
|
MgCl2 |
0.8µl |
1.0µl |
1.2µl |
0.8µl |
1.0µl |
1.2µl |
|
10X Buffer |
1.5µl |
1.5µl |
1.5µl |
1.5µl |
1.5µl |
1.5µl |
|
dNTPs |
1.2µl |
1.2µl |
1.2µl |
1.2µl |
1,2µl |
1.2µl |
|
Primer 1 F |
0.12µl |
0.12µl |
0.12µl |
0.20µl |
0.20µl |
0.20µl |
|
Primer 1 R |
0.12µl |
0.12µl |
0.12µl |
0.20µl |
0.20µl |
0.20µl |
|
Primer 1 F |
0.12µl |
0.12µl |
0.12µl |
0.20µl |
0.20µl |
0.20µl |
|
Primer 1 R |
0.12µl |
0.12µl |
0.12µl |
0.20µl |
0.20µl |
0.20µl |
|
taq |
0.1µl |
0.1µl |
0.1µl |
0.20µl |
0.20µl |
0.20µl |
|
DNA |
5.0µl |
5.0µl |
5.0µl |
5.0µl |
5.0µl |
5.0µl |
|
Total Volume |
15µl |
15µl |
15µl |
15µl |
15µl |
15µl |
