Protocol 17919: Standard PCR Assay - Fxn<em#Lutzy>-cKO
Version 1.0

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 = 244 bp
Heterozygote = 198 bp and 244 bp
Wild type = 198 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
26219 GTA GAC GTG GGG ATC AGT CC Forward A
26220 CGC TCT GCT TTT TGA TGT TC Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
26219 0.50 uM
26220 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
029100 B6.Cg-Fxnem2.1Lutzy Tg(Ckmm-cre)5Khn/J
029720 B6.Cg-Fxnem2Lutzy Fxnem2.1Lutzy Tg(Ckmm-cre)5Khn/J
028520 C57BL/6J-Fxnem2Lutzy/J
3 strains use this protocol