Protocol 43936: Probe Assay - Eml3<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

>chr19:8935165+8935274 110bp TCTCAGCTTTTGTTTCTCCATC GGTTTGACCCTCAACACTGC

Mut= 108 bp

Wt= 110 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TCTTTCTTTTATTCTTTCTTTCTTTCTCTCTCTCTCTCTTTCTCTCTCTCTCTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCCTTCTTTCCTTCCCCCCCCACCTCTCTTTCAACTAAAAGTCCCCATCTCTCTAGAGCCCTAACAGTGACCCCACattagcaggctgactgggaggaacctcctgtcagtatctttttttgttttgttttgttttttgtttttggttcgagacagggtttttctgtgtagccctggctgtcctgtaactcacttggtagactcagaaatccgcctgcttctgcctcccgagtgttgggattaaaggcatgcgccaccatgcccggctcctgtcagtatcttgtccccacttctctcaggtcttgacacttgatcctggtcttatacttttacctcttctcaatagccgggggaacaaagaccctctgtccagtcctgggggtcccggatctcggaggagcaattataacttaggtatgtaacagagaagtggagggaagaactcgcatgatggaattagtcaggtacggtgggtttgctgctctggccttcaggagggaggcttatttccctgctttttcctgtagaaggcatttcagtgaagatgttccttcgtggccgtcccatcaccatgtacattccgtctggcatccgaagtcttgaggagctgcccagcggcccgcccccagagaccctgagcctcgactgggtgtatcctattcagttccgtgggaatcacccctgcccacaatgggtccctttccttctttacagtttctttctgtgtgggggtggacaaggaatcctgtgaattatcccataccaccctaatgtggatggtgatgccaagtgggagctttgccgttttcatgttcccttcttcctcttcctgtccttcatctggccctttccttgaccatgcccctcaacacagttatgggtacaggggtcgtgattctcgctctaatctatttgtgctgcgctctggggaggtggtctactttattgcctgtgtagtggtgctgtaccggcctgggggaggcccagggggtcctggaggtggcggccagagacattaccggggtcacacagactgcgttcgatggtgagggtcttggggtctggggctggtgggcttgacaggaagagggtctgtctacggggggaagggtagcaaagacagctgccttgctgcttcttgggtggtcttgagagagacccctttcctatagttctcttccccttccttagccttgctgttcaccctgatggtgttcgtgtagcctcaggacagacagctggagtggacaaggatggaaaggtaatgtcaagcagaatatcgagcagataggatgagataacaggtcagcttttcttcccggttctgaccccttctcttccactagcccctgcagcccgtggttcatatctgggactcagagacactcctaaaactgcaggagattggactgggtgcctttgagcgaggtgtgggggccttggccttttcagcagcggtgagctgagcacaaagcttccgatcttgtttgtttgtttgttgttgttttgttttttgggacagggttcctctatgtgtagccttagctgtcctggggctcagtttgtagaccaggttggctttgttttgttttgttttttcgagacagggtttctctgtgcagccctggctgtcctggaactctctgtggaccaggctggcctggaactcagaaatccgcctgcctctgcctcccaagtgctgggattaaagatgtgcgccaccacatccgactgtttgtttttgaggcagggtctcatgtaccttcggctggcctctgtaactcaatatgtagatgaggatgtttttgaactcttgctccttctgtctctgcccaccaattgttataggcacgtgccaccatatctggctagacccttattcttgctcaggacaccactctttgattttaccaggaagcctggggaaggtctagtgttctgggctcacacagccagttctcctgagaaagagaccctcccaacctgtgatagatccctgtctcatagcaggcatgtacagacgtcccggcttcacatgctgtgtctcgtcctcaggatcaaggtgcttttctgtgtgtggtggatgattccaatgagcacatgctgtcagtgtgggactgtagccggggagtgaagctggctgagattaaggtgaggaacctaggtggcttgatggtttctggatctggggtagggtgggagtggtggaatacagacagttccaggtttacttatacatacacttccgactcagacaaactctcagttctctcagcttttgtttctccatctgtgcattgggaactacaggagttctaacGGGGACTATAATTATTTAGGGGATCAGCTCCCCCCCCCTGCAGTGTTGAGGGTCAAACCAGGGCCTTTAATATGTCAGTAGGCATTCTACCATTTGCTCTATACCTTCAGCTTTCAACCAGAGCCTTGAAGGAAGAATTATCTTGAGGTCTCCGGGCTTTTGTAGACCAGCTGTTAGGAAGATTGAGTTATAGAGTAGACTTTGGTGGCATAAGGCTATCATGCCA

 

This mutation is a 2241 bp deletion beginning at Chromosome 19 position 8,932,972 bp and ending after 8,935,212 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64395 TCT CAG CTT TTG TTT CTC CAT C Wild type Forward A
64396 GGT TTG ACC CTC AAC ACT GC Common A
64397 TCA ACT AAA AGT CCC CAT CTC TC Mutant Forward A
64398 Fluorophore-1 CAT TGG GAA CTA CAG GAG TTC TAA C Quencher-1 WT Probe
64399 Fluorophore-2 AGC CCT AAC AGT GAC CCC AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64395 0.40 uM
64396 0.40 uM
64397 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.