Genotyping Protocols Database
Stock No: 002385
Protocol 25021: Standard PCR Assay - Fyn<tm1Sor>
Version 4.2

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 = 190 bp
Heterozygote = 190 bp and 415 bp
Wild type =  415 bp
PCR products separated by gel electrophoresis on a 1.0% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10207 AGG CCC AAG TTG ACT ATC CA Wild type Forward A
10208 GGC AGC CTG TCT CAA AAG TC Common A
oIMR9535 GGG AGG ATT GGG AAG ACA AT Mutant A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10207 0.50 uM
10208 0.50 uM
oIMR9535 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
002271 129-Fyntm1Sor/J
002385 B6;129S7-Fyntm1Sor/J
012468 B6.129S7-Fyntm1Sor/J
3 strains use this protocol