Protocol 25513: Standard PCR Assay - Lrrk1<tm1.1Mjff>
Version 1.2

Notes

This assay will not work without the use of Hotstart Taq Polymerase.
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 = 220 bp
Heterozygote = 220 bp and 368 bp
Wild type = 368 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12041 TCA CTG GAA GGC AAG GAC AGT TGG Common A
12042 CCA ACA CAT CCA ACC TTT TCT TTG C Mutant Reverse A
12043 CAG GAT GCT TCA CAG ACA GG Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
12041 0.50 uM
12042 0.50 uM
12043 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
016120 C57BL/6-Lrrk1tm1.1Mjff/J
016122 C57BL/6N-Lrrk1tm1.1Mjff Lrrk2tm1.1Mjff/J
022880 FVB.Cg-Pde6b+ Lrrk1tm1.1Mjff Tyrc-ch/J
3 strains use this protocol