Stock No: 012261
Protocol 23827: Standard PCR Assay - C3<tm1Hrc>
Version 1.2

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 = X bp
Heterozygote = X bp and Y bp
Wild type = Y bp

Sequence

TCGTCCTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCC
CCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTC
ATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAAT
GGCCGATCCCATATTGGCTGCAGGGTCGCTCGGTGTTCGAGGCCACACGCGTCACCTTAATA
TGCGAAGTGGACCTGGGACCGCGCCGCCCCGACTGCATCTGCGTGTTCGAATTCGCCAATG
ACAAGACGCTGGGCGGGGTTTGCTCGACATTGGGTGGAAACATTCCAGGCCTGGGTGGAGA
GGCTTTTTGCTTCCTCTTGCAAAACCACACTGCTCGACATTGGGTGGAAACATTCCAGGCCT
GGGTGGAGAGGCTTTTTGCTTCCTCTTG:AAAACCACACTGCTCG:CCAGCTCCCCATTAGCT
CTGGGGATC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10756 AGG GAC CAG CCC AGG TTC AG Wild type Forward A
10757 GAT CCC CAG AGC TAA TG Common A
oIMR0157 TCG TCC TGC AGT TCA TTC AG Mutant A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10756 0.50 uM
10757 0.50 uM
oIMR0157 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
012261 CBA.129S4(B6)-C3tm1Hrc/Mmjax
012301 D2.129S4(B6)-C3tm1Hrc/Mmjax
2 strains use this protocol