Stock No: 002270
Protocol 22439: Standard PCR Assay - Fn1<tm1Hyn>
Version 1.3

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 = 191 bp
Heterozygote = 191 bp and 271 bp
Wild type = 271 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

Sequence

This strain has two working primer sets designed at both the 3 and 5 prime ends. The other primer set is archived.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7410 ACT CTC TCC CAG GCT CTT CC Common A
oIMR7411 CAT TGC GTC ACC TCT GAG AA Wild type Reverse A
oIMR7412 ATT AAG GGC CAG CTC ATT CC Mutant A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR7410 0.50 uM
oIMR7411 0.50 uM
oIMR7412 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
008444 129S4.129S2(B6)-Fn1tm1Hyn/J
008445 B6.129S-Fn1tm1Hyn/2J
002270 B6.129S-Fn1tm1Hyn/J
008443 D2.129S2(Cg)-Fn1tm1Hyn/J
4 strains use this protocol