Stock No: 004290
Protocol 22618: Standard PCR Assay - Ihh<tm1Amc>
Version 1.3

Notes

This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).

Use Perfect Match© (Stratagene) added before Taq to delete non-specific bands.
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 = 307 bp
Heterozygote = 190 bp and 307 bp
Wild type = 190 bp
Separated by gel electrophoresis on a 3.0% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1702 CGC ATG GAG TCC CCA AGA Wild type A
oIMR1703 CCG GCA CCA GAA GCA GC Wild type A
oIMR1704 AGG AGG CAG GGA CAT GGA TAG GGT G Mutant A
oIMR9492 TAC CGG TGG ATG TGG AAT GTG TGC G Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1702 0.50 uM
oIMR1703 0.50 uM
oIMR1704 0.50 uM
oIMR9492 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.