Protocol 28969: Standard PCR Assay - Park2<tm1Shn>
Version 3.3

Notes

Melting curve analysis is done using a Roche Light Cycler 480.
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

HET = 250 bp and 500 bp
MUT = 500 bp
WT = 250 bp

 

Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7026 CCT ACA CAG AAC TGT GAC CTG G Common A
oIMR7027 GCA GAA TTA CAG CAG TTA CCT GG Wild type Forward A
oIMR7028 ATG TTG CCG TCC TCC TTG AAG TCG Mutant A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR7026 0.50 uM
oIMR7027 0.50 uM
oIMR7028 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

Stock Number Strain Name
006582 B6.129S4-Prkntm1Shn/J
023968 B6.Cg-Park7tm1Shn Gpx1tm1Ysh Prkntm1Shn/MgoldJ
2 strains use this protocol