Protocol 23807: Standard PCR Assay - Ppargc1a<sup>tm1Brsp</sup>
Version 2.2

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant = 400 bp
Heterozygote = 400 bp and 648 bp
Wild type = 648 bp
Separate on gel electrophoresis on 1.5% agarose gel

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
8039 CCA GTT TCT TCA TTG GTG TG Wild type Forward A
8040 ACC TGT CTT TGC CTA TGA TTC Wild type Reverse A
8041 TCC AGT AGG CAG AGA TTT ATG AC Mutant Forward A
8042 CCA ACT GTC TAT AAT TCC AGT TC Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
8039 0.50 uM
8040 0.50 uM
8041 0.50 uM
8042 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.