Stock No: 018295
Protocol 23416: Standard PCR Assay - Nos3<tm1Unc>
Version 3.2

Notes

This assay does not work well without the use of a Hotstart.

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

HET = ~300 bp and 337 bp
MUT = ~300 bp
WT = 337 bp

Separated by gel electrophoresis on a 1.5% gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8963 AAT TCG CCA ATG ACA AGA CG Mutant A
oIMR9357 AGG GGA ACA AGC CCA GTA GT Wild type Forward A
oIMR9358 CTT GTC CCC TAG GCA CCT CT Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR8963 0.50 uM
oIMR9357 0.50 uM
oIMR9358 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
008286 129S6.129P2(B6)-Nos3tm1Unc/J
002684 B6.129P2-Nos3tm1Unc/J
018295 BKS.129P2(Cg)-Nos3tm1Unc/J
008340 BKS.Cg-Leprdb Nos3tm1Unc/RhrsJ
007073 CByJ.129P2(B6)-Nos3tm1Unc/J
5 strains use this protocol