Protocol 34423: Probe Assay - Tas2r103<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

>chr6:133036136-133036234 99bp AGGCAAGCCTCTCTTTCTGTG AATGTGGAAAAGGATCCCTGA

Mut= 109 bp

Wt= 99 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTTCTGAAAGTGATTATGTTAAGAATCAGACACAGCCGTCTAGAAGATTGTATTAACACATCTTTGGTAGTTCAGAAGAAATTAGATCATCATGGTGTTGACAATAAGGGCTATTTTATGGGTAACATTGATAACTATTATAAGTCTGGAGTTTATcataggaattttaggaaatgtattcatagctctcgtgaacatcatagactgggttaaaagaggaaagatctctgcagtggataagacctatatggccctggccatctccaggactgcttttttattgtcactaatcacagggttcttggtatcattattggacccagctttattgggaatgagaacgatggtaaggctccttactatttcctggatggtgaccaatcatttcagtgtctggtttgcaacatgcctcagtatcttttattttctcaagatagctaatttctcaaattctattttccttgttctcaaatgggaagctaaaaaagtggtatcagtgacattggtggtatctgtgataatcttgatcatgaacattatagtcataaacaaattcactgacagacttcaagtaaacacactccagaactgtagtacaagtaacactttaaaagattatgggctctttttattcattagcactgggtttacactcaccccattcgctgtgtctttgacaatgtttcttctgctcatcttctccctgtggagacatctgaagaatatgtgtcacagtgccacaggctccagagatgtcagcacagtggcccacataaaaggcttgcaaactgtggtaaccttcctgttactatatactgcttttgttatgtcacttctttcagagtctttgaatattaacattcaacatacaaatcttctttctcattttttacggagtataggagtagcttttcccacaggccactcctgtgtactgattcttggaaacagtaagctgaggcaagcctctctttctgtgatattgtggctgaggtataagtacaaacatatagagaaTTGGGGCCCCTAAATCATATCAGGGATCCTTTTCCACATTCTAGAAAAAAATCAGTTAATAAGAACAGGAATTTAGGAAGGAATCTGAAATTATGAATCTCATAGGCCATGAACCTTCAGACAAAGGATTCATTAGAGAGATAGAGAGAGAACATTG

This mutation is a 861 bp bp deletion beginning at Chromosome 6 position 133,036,176 bp and ending after 133,037,036 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43578 AGG CAA GCC TCT CTT TCT GTG Wild type Forward A
43579 AAT GTG GAA AAG GAT CCC TGA Common A
43580 CAT CAT GGT GTT GAC AAT AAG G Mutant Forward A
43581 Fluorophore-1 TGT GGC TGA GGT ATA AGT ACA AAC ATA Quencher-1 WT Probe
43582 Fluorophore-2 AAG TCT GGA GTT TAT TTG GGG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43578 0.40 uM
43579 0.40 uM
43580 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.