Stock No: 010685
Protocol 23548: Standard PCR Assay - Adora2a<tm1Jfc>
Version 1.2

Notes

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

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 = ~550 bp
Heterozygote = ~550 bp and 364 bp
Wild type = 364 bp
 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9055 GGG CTC CTC GGT GTA CAT Common A
9056 CCC ACA GAT CTA GCC TTA Wild type Reverse A
oIMR6393 CAT TTG TCA CGT CCT GCA CGA C 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
9055 0.50 uM
9056 0.50 uM
oIMR6393 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
010685 C;129S-Adora2atm1Jfc/J
034539 STOCK Adora1tm1Jgsc Adora3tm1Jbsn Adora2atm1Jfc Adora2btm1Till/ReiMmjax
2 strains use this protocol