Protocol 34610: Probe Assay - Nufip2<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

>chr11:77691431+77691566 136bp ACAACAAACCCTACAATACAGCT GCATTACCGTTTATTTCACCATAG

Mut= 136 bp

Wt= 136 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCTACTGGCCCAGAAGATGGACCTGATATGAGTTGTGTCCTAGGTCTGCCTCTGCCTAGACATTGAACCCTGTATCATAATCTGTAAAAGTTTCTTTACATAAAGTTTCTTTACATAAAGATAAGATACAACAAACCCTACAATACAGCTTAgtattgggttcagtaaagttgtttggtcttagtactactgtgatttaaatgtatgtgttttgtttgtttttgttttccttctataggctatggtgaaataaacggtaatgctggagaaagagaaatatctttaaagagcctcagttctgatgaagctaccaaccctatttccagggtccttaatggcaaccagcaagttgtagaaacaagcctgaagcagactgtaaagaccagcacctttggaaaagcaggaatcaaaaccaagaattttattcagaaaaacagtatggacaaaaagaatgggaagtcttatgaaaacaaatctggagagacccaggctgtagataagaccgataccatagcaattccaaatggtgttataacaagtagttcaggctatattactaatggttatatgagcaaaggagcagataatgatggtagtggatctgagagtggatataccactcctaaaaaaaggaaagctaggcgcaatagtgccaagggttgtgaaaaccttaatttagtgcaggacaaaataatgcaagagactagtgtcccagcattaaaacagggacttgaaactttaaagcctgactatagtgaacaaaagggaatgcgagtagatggttcaaagcctatttggaaatatgaaactggacctggaggaacaagtcgtggaaaacctgctatgggtgacgtgcttcgtaaaagctctgatattaaacctggtttgagtagcaaaaaatttgatgatcggcctaaaggaaagcatgcctcagctgctgcctccaaagaggactcatggaccttgtttaagccacccccagtttttccagtggacaatagcagtgcaaaaatagttcctaaaataagttatgcaagcaaagttaaggaaaacctcaacaaaactgtacagaactcctccgtgtcaccatcttcatcttcatcatcatcatctactggggaaactcagacccagtcttcaagtcggttatcccaggtcccgatgtcagctctgaaatctgttacttctgccagtttttctaatgggcctgttttagcaggaactgatggaagtgtgtacccatctgggggtcagccactgctaacgactgctgctaatactttaacacccatctctactgggactgattctgttcttcaggacatgagtctggcttcagcagccgttgaacaaattaaatctagcctttttatctacccttcaaatatgcaaacagtgctgttaagtgcacaagtagatctgccctctcagacagatcagcaaaacctgggggatatcttccagaatcaatggggtttatcatttataaatgagcccagtgctggcccagagactgttattggaaagtcatcagatcataaagtgatggaagtgacatttcaaggagaatatcctgccactttggtttcacagggtgctgaaataattccctcaggaactgagcatcctgtgtttcccaaggcttatgagctggaaaaacggactagtcctcaagttctgggtcacattctaaaacctgggactactgagagtggagccttatccttggaccccagtcatataggtgacctgcaaaaagcagacaccagtagtcaaggtgctttagtgtttctctcaaaggactacgaaatagaaaatcaaaatcccctggcctctcctacgaacactttgttaggctccgccaaagaacagagataccagagaggcctagaaaggaatgatagctggggttcttttgacctgagggctgctattgtatatcacactaaaggtaactcatttgttttttttctaagattccaatgggctgattgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtacatgtatatttgatattctatattatgaatgactgggtaagaattagtatcaattaattgttaattgatttgaaatttttgaaaatttattaaagtttagaggactgtagttttccaacgtaaaatcatcaataaaggcacagttgatcacataattagatagaggtttcatgtcccttaaaggatagcctttttttgtaTTGGGTCTCAGGTTTTTTTTTTTTTTTTTCTTTCTTTTTTTTAAGTGATAGAGTCAAAGTTTTAAATTACCCAAATGGGCTTTAAATCCTGATCCTCTACATACTAGTTGCTGCTGGGATTGCCTGTGTATGCCACCAAA

This mutation is a 2080 bp deletion beginning at Chromosome 11 position 77,691,456 bp and ending after 77,693,535 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44010 ACA ACA AAC CCT ACA ATA CAG CT Common A
44011 GCA TTA CCG TTT ATT TCA CCA TAG Wild type Reverse A
44012 GCA ACT AGT ATG TAG AGG ATC AGG A Mutant Reverse A
44013 Fluorophore-1 ATT GGG TTC AGT AAA GTT GTT TGG Quencher-1 WT Probe
44014 Fluorophore-2 AAG TTT TAA ATT ACC CAA ATG GGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44010 0.40 uM
44011 0.40 uM
44012 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.