Stock No: 017835
Protocol 26592: Standard PCR Assay - Brca1<tm1Aash>
Version 2.2

Notes

Melting curve analysis is done using a Roche Light Cycler 480.

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

Melt Peaks

Wt = 85.6°C +/- 1
Mut = 81.3
°C +/- 1

Mut = 210 bp
Het = 210 bp and 442 bp
WT = 442 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10609 GTC AGC AAA GCC CGG GTT TGG T Mutant A
10807 AGG GCC ATG ATT GTC AGT TC Common A
10808 GAT GGA AGC TCC TTC ACC AC Wild type A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10609 0.50 uM
10807 0.50 uM
10808 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.