Stock No: 028785
Protocol 19348: End Point Analysis Assay - Grin2a<em#(S664G)Frk>
Version 1.0

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 = G/G
Heterozygote = A/G
Wild type = A/A

Sequence

TCTGTGCCCGTCCAGAATCCTAAAGGCACAACCAGCAAGATAATGGTATCAGTGTGGGCCTTCTTTGCCGTCATCTTCCTTGCA(a/g)GTTACACA
GCCAACCTGGCTGCCTTCATGATCCAGGAGGAGTTTGTGGACCAAGTGACTGGCCTCAGTGACAAGAAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
28526 ATC AGT GTG GGC CTT CTT TG Forward A
28527 TGG TCC ACA AAC TCC TCC TG Reverse A
28528 Fluorophore-1 TCT TCC TTG CAA GTT ACA CAG CC Quencher-1 WT Probe
28529 Fluorophore-2 TCT TCC TTG CAG GTT ACA CAG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
28526 0.40 uM
28527 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
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.