Protocol 47282: Probe Assay - Tmem185b<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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=  98 bp

Wild Type = 90 bp

>chr1:119526483+119526572 90bp CCCATCTAGAGGCAGTACA AAGCAGGCATAAATGAGGAA

Sequence

Large deletion:  Mutant sequence with junction in uppercase

ccgggccacgtgactgctcgcacgtgttccggcccctcggagctgcggtctggagcctctaacacgcagcgggcggatgcccatctagaggcagtacagccttcgccATgatatgccagactaaactcacaggctgggcctttcagtgaaataagaaccatatacccttgaattccactttgcttttttcttgtgttcatccagtcctggaattggaaacaggtccccctc

This mutation is a 1,036 bp deletion of Chr1:119,526,512-119,527,547 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
70193 CCC ATC TAG AGG CAG TAC A Common A
70194 AAG CAG GCA TAA ATG AGG AA Wild type Reverse A
70195 TGG AAT TCA AGG GTA TAT GGT Mutant Reverse A
70196 Fluorophore-1 CTG TTC CAG GAC TTC AAC C Quencher-1 WT Probe
70197 Fluorophore-2 CCT TCG CCA TGA TAT GCC AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
70193 0.40 uM
70194 0.40 uM
70195 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.