Stock No: 003812
Protocol 22291: Separated PCR Assay - Hfe<tm1Gfn>
Version 1.3

Notes

Please note attached gel image shows wild type amplification products in the first row and mutant amplification products in the second row.  The primer numbers used for the mutant products are 1352 and 7595 (NOT 1352 and 1354 as indicated in the gel image).

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 = ~250 bp
Heterozygote = 333 bp and ~250 bp
Wild type = 333 bp

<p>Separated by gel electrophoresis on a 3.0% agarose gel.</p> <p>Please note attached gel image shows wild type amplification products in the first row and mutant amplification products in the second row.&nbsp; The primer numbers used for the mutant products are 1352 and 7595 (NOT 1352 and 1354 as indicated in the gel image).</p>

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1352 GAA TTA ACA GGC CGT TTC TAA AG Common A, B
oIMR1353 CTT GGA GTA GTG GCT CAC ACT Wild type Reverse B
oIMR7595 GAG ATC AGC AGC CTC TGT TCC Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR1352 0.50 uM
oIMR7595 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.

Reaction B

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR1352 0.50 uM
oIMR1353 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

This is the only strain that uses this protocol.