Protocol 44062: Probe Assay - Prr30<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

>chr14:101199487-101199588 102bp GAGGGTCTTGAGGTCCCACT TGCGGAGGAAAAGATTTTGA

Mut= 94 bp

Wt= 102 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TGGGATTGCAGGCTCATGCCAGCATGTCATGCTTATGTGGTGTTGGGTATCATACCAAGGACTTCACGAATGCTTGGCAAACAATCTACCTGCTGAACAACAGCCAAAGACCTCACATGGAGGGTCTTGAGGTCCCACTAGAAATGAGCACCTGAAGCAGAAtccaggagtgggccaatcatccctacattttgacctctgtcaaaatcttttcctccgcaggtcctacaggctgtgtcttcctcctgattccatccagatagttgtggttgtcccttagctcctcaggaacagccctcatcctctgtttccgtacgcactgcacctccagcgaatggctaaggagacgtgaggagccaagcttagatgcaggctctggactgagcacgccacagccaggtagaagaggctgcgtgtcctcttggcctgctgctgatgcctgcagctgttgaggccatactgtgcacagaaggcagtcttgaccaccagccacagctggcctcagagatctcccacgctgactgttgggtccctaaccactggtcttgtccaccccatccacttccctcctggattcaaggcaatgttgcctgtaaacaaggaccaggtgctgctacagaacacagtacctccagggtgtccccctcaggtcctctcacagtttgtgaattcccctgcccccaacttagcatctctttgtccccatccgaccctcccttcttcccactttcctctgcctgcccccccacaggcttatttcttctcttctttgactcagactcactctcctgggcctcacttctcttcggactcaaattctgactttgttcctcctcactcttctagtcacccacgatcctcttcttgctttggccaaaactatacttattttggtgaaaaactcccctctccacactccatctctccctctaactatcagctctgtgtgtctccccctctcaccggctcctcctctctttcccagctacaacactcctctcctcactcctgtcagtccccttcccgccttcaggacttacaaagccccaagatcacttccccggtccccagctcaccttctccaaggattcagaataacaagcaaacatggcagtggcctcagtccggaagcatcaagtcttctcggggggcaggggtatgtgtgccaagcaaggtggaccctgcagaattcaaggactcagggaccctcacccaagccctggtggaccatgtggggcgccgcagaattgcacgagacctgcagatacagtttctgcagcgcctgtggctcggcacacccggccatgccccagtcgtggagtaccccatatgtctagtgtgcctccagatccgcaccccatcttgtcccacccccaagtacaagactgtaccccagctgcttgccttccctcaactactaccctgtgtccagggccaggaatctgggcctctccgaataggaattggcttcggcctccgcctgcctcggggccaggccagggctttgcatctgctgcccccaaaaaactctactccggtgggagtagagtctcaggaagaggccctccagcgacaaaaatccacaatccaagaatcagttcaaatcaccggcacactcttccaggcccggagcctccggtctatagaccttcaatcaccaaaacccagccagtgttccaggtctctacttcaggaaccaagacaggtcgctgcatccccaaaggccgggccttctgtctcaaagaggtctgtgactctagggtctattctccgaaagtcgccatcttaatcccagtgcagcgaagaccctctggaacaccacctccaaactctaccttccctaccaggacttctcagaccttggagggctggctggctggaagccaggtgtgttctcctgccctgaaccctgggaaccccttgtggagtttgctctcccctagttgaagagaccagaggcacccaaggcctcatttagccttcagtgtatccaataaagcctaactccgagaatcttatgtttgtgtctactatgcccacaggatggaatcaaggctatgggataaagttagtccgacaaaagacttggcgggggggcgggggggcgcggagaaagattaaggataccaactGCTATCACTTGAGTACATGCTAGTACCTGATGCTTAGAGACCTCCACAAGGCCCTTCTCCTGTTTAACTCAAGTCTGTGGACAGATAGTTGGAATCAAAGATTAGGCCAATGGGTCTATTTCTCCAGGGACAGCCTGGGTTGACCAGGATGCAGCAGCTCATTTTCCTCACAGGGAAGGACATTGT

 

This mutation is a 1970 bp deletion beginning at Chromosome 14 position 101,435,012 bp and ending after 101,436,981 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64695 GAG GGT CTT GAG GTC CCA CT Common A
64696 TGC GGA GGA AAA GAT TTT GA Wild type Forward A
64697 CCT TGT GGA GGT CTC TAA GCA Mutant Reverse A
64698 Fluorophore-1 CAA TCA TCC CTA CAT TTT GAC CTC Quencher-1 WT Probe
64699 Fluorophore-2 CCT GAA GCA GAA GCT ATC ACT TGA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64695 0.40 uM
64696 0.40 uM
64697 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.