Protocol 35400: Probe Assay - S100a16<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

>chr3:90543152+90543251 100bp CTCTTGGCTGCTTCCTTTGT GCAAGTCCCTACAGCCTCAG

Mutant=  97 bp

Wild Type = 100 bp

Sequence

AGCCCCTGCTTTCCTCTGTCTTGATTCTTCCCAGCTAAGGTTGCCACAGAACAAGGGACCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAGGGGGCAACAGTTACCTGCTTCCTCTCCTCACAGGAGCCTCCTGCTCTCAGAGGGCTCTGACAGGCATCAGGCCTGCCTGGGGCAGGGGAAGGGCCTGGAGAACCTGTCTTGgcagttggcagtctggctggggaacacctggattagaggagtcacatagctctccttccaggccacccccacacccactttgctatcctatgtctgctttctcccttctctgaaaggcaagaagtctgaggatgctgtctgtgtgcagggatgtcccttctcatctctcttttctgcttcctaggcccagagaggagctgaggcagtgagatggctgactgctatacagagctggagaaggcagttgttgtcctggtggaaaacttttataaatatgtatccaagcacagcctggtcaaaaacaagatcagcaagtccagcttccgaaagatgctccagagagagttgaaccacatgctgacggtatccccactgctgccctctgtgcctgctcttctcctcacagctctctcagtgacccctccctccctgtgctctgctccgtgttccctcctagcctgctagcctcctctgccctgctagcctggccaagggactctgcctagggtcctgtctgggccatcccaagctttactgattcaagttcttccacaggacacagggaaccgaaaggcagctgacaagctcatccagaacctggatgccaaccacgacgggcgcatctgctttgacgaatactggaccatgattggtggaatcaccagccccatggccaacctcatccgccaacaggagtgccagcaggagagccagcaggagtgccagcaggagagccagcaggagagccagcaggagagccagcagggcagcagctagaggcccctcgtgggccccttttcatgtcctgacccatgtcctgccagtgttctttccatgctttaagttgactctttcttcctcccttctccctccagacctgttcctacccttgctgaacagcacaagatgggtgggtcgggggctatcaaggccactgtggtagtgcttccctgagcattcaatccctggatcctgtaggccctgggagggggtcccctttgtgaaatatcaatgctgtttggggccctccccccaagacttctctcacttgttgagtctgtgtgctctcgcaatgttcctgtctaattcttgactgattctgggtctgtcctgacctctagaggtgagcttgctgcttgaggtctcccagctcctgactgtggcagctgtggtagtggcagtaccagaattagccgcttcttttcctcccaacctcttagccgggtagagaggcattcccaggggctcctgtccagctgtctctgtctgggaatgatctaacctgggttgagcctcctctctgctctgccaccgaaataaacgttgaatttggagtttgtaactcttggctgcttcctttgtctttaattgctccacacttcttgggaatctattctaccttggtgATCTTTGTCCTGGACCCTGAGGCTGTAGGGACTTGCATAAGAAAACATTACATGCAAACAAAATAAGAAGAAGTTAAACTGGGATTGATGGCCCGCATGTATAATTCTTGAATCTAGGCTGATGTATTAGGGGTCTGGAGTTCAAGGCCAGCCAGAGATGCATAGCTCAGCATGGTGGCGTTTGCCTGCCCAAAGGCAAAAAGAATAGGTTATATTCTCAGATTCAAGTTCAACTTTTAGGTGCATAGTAGGTGT

 

A 1406 bp deletion beginning at Chromosome 3 position 90,541,810 bp and ending after 90,543,215 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45960 AGG GCT CTG ACA GGC ATC AG Mutant Forward A
45961 CTC TTG GCT GCT TCC TTT GT Wild type Forward A
45962 GCA AGT CCC TAC AGC CTC AG Common A
45963 Fluorophore-1 TGC TCC ACA CTT CTT GGG A Quencher-1 WT Probe
45964 Fluorophore-2 AGA ACC TGT CTT GAT CTT TGT CCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45960 0.40 uM
45961 0.40 uM
45962 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.