Leprosy Research Support - STR Primer Set

STR Primer Set

Hints and Tips:

Please use the enclosed standard genomic DNA 4264 as control template for relative sizing of the minisatellites amplified from the test samples.
10-20 ng in a 25 µl reaction
The PCR fragments are resolved in a 3% agarose gel with 1X TBE buffer. We don't run more than 8 lanes so that bands are straight and fragments in one lane can be compared to another.
We run the 20 bp BIORAD EZload DNA ruler in the first and last lane to ensure a “straight run.”
The following are the conditions at which we amplify the loci:

Platinum PCR Supermix (Invitrogen Corporation, CA): (contains Platinum Taq polymerase, dNTPs and buffer)	22 µl
Forward primer (from 10 µM stock):	1 µl
Reverse primer (from 10 µM stock) :	1 µl
M. leprae standard genomic DNA (4264, 20 ng/µl) :	1 µl
*Or test DNA*	Up to 3 µl
Total 25 µl
Note primers are at 400nM final concentration. They can be reduced to 200 nM by diluting the starting stock.

PCR Program

Activation step at 94°C for 2 minutes.

First 10 cycles:

Touch down PCR

Denaturation:

94°C, 30 sec

Annealing:

65°C to 55°C (Temperature reduced at the rate of 1°C per cycle), 30 sec

Extension:

72°C, 30 sec

Followed by: 25 additional cycles at fixed annealing temperature of 55°C

Denaturation:

94°C, 30 sec:

Annealing:

55°C, 30 sec

Extension

72°C, 30 sec

Final Termination:

72°C for 5 minutes

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