Stock No: 002913
Protocol 22844: Standard PCR Assay - Gria2<tm1Rod>
Version 2.3

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 = 164 bp
Heterozygote = 164 bp and 361 bp
Wild type = 361 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
oIMR6780 GGT TGG TCA CTC ACC TGC TT Wild type Forward A
oIMR6781 TCG CCC ATT TTC CCA TAT AC Common A
oIMR8444 GCC TGA AGA ACG AGA TCA GC Mutant A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR6780 0.50 uM
oIMR6781 0.50 uM
oIMR8444 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
003143 B6.129-Gria2tm1Rod/J
002913 STOCK Gria2tm1Rod/J
2 strains use this protocol