Protocol 30113: Probe Assay - Hmg20a<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr9:56321958+56322068 111bp TGCTCGTTAAGAAATGGCTCT TGCTACCTTCCATGTGTTTCC

Mutant=  115 bp

Wild Type = 111 bp

Sequence

Wt Sequence: tgctcgttaagaaatggctcttatatgatgagtcaaagacctggacaaccccattcaTCtggtccattactagggtttccacatcacatgggaaacacatggaaggtagca

 

Mutant Sequence: tgctcgttaagaaatggctcttatatgatgagtcaaagacctggacaaccccattcaTCaggtaactcaagctgctagaattgcagatgtgctcaccatgctcagtcgtgacaaa

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
33931 TGC TCG TTA AGA AAT GGC TCT Common A
33932 TTT GTC ACG ACT GAG CAT GG Mutant Reverse A
33933 TGC TAC CTT CCA TGT GTT TCC Wild type Reverse A
33934 Fluorophore-1 CTG GTC CAT TAC TAG GGT TTC C Quencher-1
33935 Fluorophore-2 CTC AAG CTG CTA GAA TTG CAG Quencher-2

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
33931 0.40 uM
33932 0.40 uM
33933 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.