Protocol 37190: Probe Assay - Rnf217<em1(IMPC)J>Alternate 1
Version 2.0

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

 


>chr10:31506063-31506185 123bp CCCAGTATGCATTTCTTAAAGC AAAAGTCTTATGAGCATCCCTGAAG

 

Mut = 127 bp

Wt = 123 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CATACCACTAACTCTGTGTACCTGAGGAGAAAAGAGTAGAGTCACAAGTGAAATTGTAGATTCTGGCAACAACTTACCTGCTAAGCATCAAACAAACTCTGGCAGGTTTGACTCTTTGTTTTAATTATAAAATTCCTAACTACATTAAAATATTCCCCAGTATGCATTTCTTAAAGCCATGTTacaggatcataatggtaaaactaatattgggaaccttataaatacgattataaacaaattgaaaacttcacttcagggatgctcataagacttttaatttggtaaaaccgatctattttacttgacatattttactttttaagcacattaattgcatttccttctatttgtccctttatccagatccatatccagagaacagaagggtgtgaccatatgacttgttcacagtgtaacactaatttttgctatcgatgtggagagagataccgccagctccggtttttcggagaccacacatcaaacctcagtatatttggatgcaaatatcgctacctcccagagagacctcatttaagaagattagttcgagggtcagtctgtggtaagtagcacacatgattgctctcaggtgcctccttaggatccgaggaagggcaatagcatgagaatatcagccatgcgaatctccagggcttgctatgggccaggggttccagtaggtcctttaccaaagagtgagaagagaatcactccttttatgaacaaaattgaaaagttaaggttgttaacatgacattctttcatgatcagcgcttctgaaaccctgggaaagccaattgctgacaagaagaatgtatggacttaacaggtatctaaacacaaggatgtgctgttgattgtttatagaatatgaaatgggacaatgatgaaaaagcacacacatcccaaagaaataaacaaataaaacagtaaatgttaagacacaatcaatggtagattgtcatagcagaaattaaacaaaattttaaattaaaaatgtttaaataagatataatttatggttgtgaaaaattatatttgattattgaaaatgatgttctaagagaattgttactgatcttggaataagagaaagcgtgcttactccactcaaaatcatctttagcagtccatgtcctacactgtcaagtttattccttctaaagggcagatatttgctccttgtattccgtgatggttgggaagaaaatgtcagtgctattattacttgacagttatattctgatgctgtgaagaaatgtcttggtggatactttaaggatatattactaaagagattaagctgtatgtttataatatgtaaccctttgtactgtattctagcttcttccacacagctctatggatacacatgcccttgttcaataagatttcatgtcacttaactgagttttgttattgtgtaaagcaaaagaaaaaaacgtatgtctgggcattatgttaaatggacaacaattcatatctaaatactaggtattaagtaatgctaaaattgatatttaaaactgtatttttttatatctagtcatctaattccaaaactgttttctatgaattaaaaaagttaatttttcctgccaaactcccaacaaatggctgaattatctttcattcattggtcaatgatcataacttatagatggtattgctgtgtgattcttaagtagctgtgtgactactgcagacttcctgttcccttcacactctttgtgacatttcgtttgtacggttatgtcaaaagtcacagcacagtaacaagaaataagtaactgtcctggttttgtctatgttggagccttttagagatatagggagtcctctagtggcttctgggctgcagttacatgtaacaatggtgattattatcacagatcaaatattcattcagcagagaaaccgctggtgctgccaagcgtgctgtgcctgccagcactgagctgtcttacaatgctgcctgttaacatttgtttcagtgattcacttgggtaacttccagcacatcacttacgcagtgggaagttcccctagcagttgtcttcaggcatcccatttggagttcagtctgttagacattcatatgatgtccctctttatctagcaagtaaataaagcattgttcttagacatggggctggtcagacaccctctctgatccctactggctgtctaactttgtgagctcaatagattatcctctgagtcactccattcttaatcgttcaacattttgtacgttgaaagtaggagaaatctaaaggaaaagcacagagactgaagcgcatcagtgaaaaggattatagtctgttttgggtataaatagtaagggacctgtggctacacggcatctgcccatggctttgtggcactgtacatacaccatgtatcagttctatatgtgcccttaattgttcttatgcttctttaaggattactgtaactgctctgtaactcagcagtgagagcctttaactcttatacttttgtttttcagctggaaagctcttcattgcgcctctcatcctggttttgggattggcactaggggccatagcagttgtaatcggtaagacacgtgacatatttgagattagatttctcccacagcagtagagaccgtgtgagtgtacatgtggtggtgtgagtcagtcagtttgtgtggtttctgctctgccttgcttaaaacagccatgcagttcagggcatataagtcagaggaaCATGTTTCCTACAGGATGTGTTTATGAGACTTTAGTATATAAACATATGTATTAAAGGAGTAGTTTTCAAACAGCATGATAGTCTGTAGTCTGTTGCAGACACACATTGCTAGATTCTACTGTCAGTTTCTAGCACAGTGTGCTGGCTGGACCCATGAGTTTACGTTTCTA

This mutation is a 2551 bp deletion beginning at Chromosome 10 position 31,503,602 bp and ending after 31,506,152 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49293 CCC AGT ATG CAT TTC TTA AAG C Common A
49294 AAA AGT CTT ATG AGC ATC CCT GAA G Wild type Reverse A
49296 Fluorophore-1 CAG GAT CAT AAT GGT AAA ACT AAT ATT GGG Quencher-1 WT Probe
49297 Fluorophore-2 TGT TCA TGT TTC CTA CAG GAT GTG TTT AT Quencher-2 MUT Probe
50767 CTG CAA CAG ACT ACA GAC TAT CAT GC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49293 0.40 uM
49294 0.40 uM
50767 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.