Stock No: 003812
Protocol 9115: Separated PCR Assay - Hfe<tm1Gfn>alternate1
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 = ~400 bp
Heterozygote = 377 bp and ~400 bp
Wild type = 377 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12834 GCT ACT TCC ATT TGT CAC GTC C Mutant Forward
15294 TCT GGG ACA GCA AGG TAT GA Wild type Forward
15295 GGC CAC TAA CAT TTC CCT TTC Common

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
Primer 1 0.50
Primer 2 0.50
10x Loading Dye 0.50
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -1.5 C per cycle decrease
4 68.0 --
5 -- Go to step 2-4, 10 times
6 94.0 --
7 50.0 --
8 72.0 --
9 -- Go to step 6-8, 28 times
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Reaction B

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00
MgCl2 2.00
dNTPS-kapa 0.20
Primer 3 0.50
Primer 4 0.50
10x Loading Dye 0.50
Kapa 2G HS taq polym 0.01
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -1.5 C per cycle decrease
4 68.0 --
5 -- Go to Step 2-4, 10 times
6 94.0 --
7 50.0 --
8 72.0 --
9 -- Go to Step 6-8, 28 times
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.