Protocol 47916: Probe Assay - P2ry10<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=  97 bp

Wild Type = 114 bp

>chrX:107103373+107103486 114bp CTATCTCGTCATGGAAGCTC GTCAGGTAAAGAGAATCCTAACT

Sequence

Wt Sequence (deletions in lower case; insertion with carrots^^):

TATTCTATATCTCAGTGTAAAAGAGAGCCCTAGATATTCACTAAAGTATTATTTAACCATTGTGCCTTTAAACTTTGTGTGTTTTTTTTTACAGGAGCCATAATTCCACAGTTTTCCTTGccaaacatactgaaatattcaA^a^GatgggcagcaacagtaccagcagtgctgagagcaattgcaatgccacttatttgccatttcagtactctctgtatgcaactacctatatcttcatattcatcccgggtctcctggctaacagtgcagccctgtgggttctgtgccgcttcatcagcaagaagaacaaggccatcattttcatgatcaacctctcagtggcagaccttgctcatatcctgtccttacctcttcggatttactattatatcaatcgtcactggccattccagagggccctttgcctgctgtgtttctatctgaaatatctcaacatgtatgccagcatttttttcttgacatgcattagccttcagaggtgcctctttctcctcaaaccattcagagccagaaactggaagcgtaggtacgatgtgggcatcagtgctgtcatctggattgttgtagggactgcctgtttgccatttcccatcctgagaaatgctggcttagccaatagcacagattcttgctttgctgatcttgggtacaaacagatggatgcagtggttctggtcaccatggttgtaattgctgaacttgctggatttgtgattccagtgatcaccattgcatgttgtacctggaaaacaactgtatccttgaaacacccaccaattgcttttcaagggattagtgagaggaaaaaagcattgcggatggttttcatgtgtgctgcagtcttcgttatctgcttcactccttatcacattaacttcattttttacaccatggtaaaggaaagcatcattaccagttgtcccactgtaaaaagtaccctgtatttccatcctttttccctatgccttgcaagtctctgttgtcttttggacccaattctgtactatttcatggcttcagagtttcgtgaccaactatctcgtcatggaagctctgttactcgttctcgtctcatgagcagggagagTGGATCATCAATGGTTAACTAAAATGAGAAAATTCTTCAGTTAGGATTCTCTTTACCTGACATTCTGTGGCATTGCCCAAAGGCAGGAATTTCCAGGCAAATAATTTCTTTAACTGAACCTTGGAA

 

This mutation is a 988 bp internal deletion of region ChrX:107,102,438-107,103,425(GRCm38/mm10) and removes most of exon ENSMUSE00000383594 and has a 1 bp insertion at ChrX:107,102,459.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
71609 CTA TCT CGT CAT GGA AGC TC Wild type Forward A
71610 GTC AGG TAA AGA GAA TCC TAA CT Common A
71611 TAC AGG AGC CAT AAT TCC AC Mutant Forward A
71612 Fluorophore-1 TGT TAC TCG TTC TCG TCT CA Quencher-1 WT Probe
71613 Fluorophore-2 TTC CTT GCA AGG TGG ATC ATC AAT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
71609 0.40 uM
71610 0.40 uM
71611 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.