Protocol 37426: Probe Assay - Slc10a3<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

>chrX:74371058-74371161 104bp AGGCAGCTGTGCTGAAAGAG TGCAAGCTTAGGAGCATGG

Mut = 101 bp

Wt = 104 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GAGGCAGCTGTGCTGAAAGAGGAGATGGCTGAATGACATTCaagggccagcattatttcaaaactagaggtaaaggtctgagagccccatgctcctaagcttgcaatttctcctcttcattaaccactttctttaccccttttcttttcaggaaaaaatcctgctatcaaggaagaaaatataggaccaggaccttcccatggacccttctcaggacattccaacagcacaagcaagggtgtctccctaagacacaagaggcctgaaaaaactatcctttgtgaagtgccatggtggtcaagaaaagcagaggtagctcccagcagttgcctggcctgggaggacagggtggttgtacaggttttgtaggcatgcttggaactgccctgctgttcatcagcctaccatggggagctcaagtgatggccagtgccaacatcagcactgctctgggccacacaagtggtcactatttgagtattggggatggctcagtgacagaatttgatttccctgagaagagtgagggtatcattgtgatctctagccaatacactggacagaccaatgggacaggccccagccccatacttaaggttatatccctggacacagaggtgctgaccattaagaatgtgagtgccataacctggagcagtggaggtggctttgtggtaggcatccactcaggtctagctgggctggccccactccacctccagctcatggatttctatgaggccccacccctgctgatcgaagaacggagagatttctgcatcagggtgtcacctgctgaagacctcccttctgctctcaacaccaacttgggccacttctcagagaacccaatactctacttgcttctgcctcttatctttgtcaacaagtgttcatttggttgcaaagtggaacttgaagttttgaaggagctcctacagagcccccaacccatgctgttgggcctcttgggccagtttctggtcatgcctttctatgctttcctgatggccaaagtcttcatgctacccaaggccttagctctgggcctcatcattacctgctcatcacctggtggtggagggagctacctcttcagtctcctccttggaggagatgtcaccctggccatctccatgactttcatctcaacagtagctgccactggtttcctaccactctcatcagccatctacagctaccttctcagcatccatgaaacactccacgtgcccatctccaagatattggggaccctgctgtttatcgccatccccatagcagcgggtgtggtgatcaaatctaagctccccaagttctctgaactactgttacaggtcatcaagcccttcagctttatacttctcctgggtggcctgttcctggcctaccacatgggggtcttcatcctagtgggagtcaggttacccattgtactggtgggtttcacagtgcctcttgttggcctcttggtgggctacagcctggccatctgcctgaagctgccagtggctcagcgacgaacagtcagcattgaggtaggggtgcaaaacagcctgctagccttagccatgctacagctgtctctgcgccgccttcaagcagactatgcctctcaggcccctttcattgtggcactgagtggtacctccgagatgttggctttggttattggccagttcatctatagcatcttgtttcctgttccctgagccctctggatcaagttttataacttccagcccccatactcctctcttcccacagttcttgtgtaccgtaccaaaggcctttagttctcatgcactatgcactcaagacaaatccaggcttatttttttactccttttcgccccagctttcagtgccaaagaggccatgctgagttagatagttgggtattgcccagaaatatatttcaataaaaaaaaaccaaaaaaacaaaagcaagctcgggtcccttccagtctgtcccttggagcctgtggattggggtaagaagagtgtgtctgggcggatatgggccagaacagtgaaccagcttggtgaccctgcacagaacccactttctcctcctcactctgttttgcacatggtagacatgtacctttgagctgtatcctcagatctacaaaggctttgctctaacagccctacctaacagggcagacctctgcACTATAAGGAGTGAAGAGAGCACTACTTCCGAGCTTCAGTCAGGATATTTTTGCAGCCAGACTAGAAAGGAAGTAGA

This mutation is a 2136 bp deletion beginning at Chromosome X position 74,368,986 bp and ending after 74,371,121 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51320 AGG CAG CTG TGC TGA AAG AG Common A
51321 TGC AAG CTT AGG AGC ATG G Wild type Reverse A
51322 TCT GGC TGC AAA AAT ATC CTG Mutant Reverse A
51323 Fluorophore-1 AGG GCC AGC ATT ATT TCA AAA C Quencher-1 WT Probe
51324 Fluorophore-2 CAT TCA CTA TAA GGA GTG AAG AGA GCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51320 0.40 uM
51321 0.40 uM
51322 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.