Protocol 36505: Probe Assay - Ipo4<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

>chr14:55632770-55632860 91bp GTGGTGGGAAAGGAATAGCC TGTATACCCCTTCTCTGCCTTC

Mut = 111 bp

Wt = 91 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GAAAACTAAAGTTTTTTGTGATTTATTTTTCATTTTTTAATTTTTTGGTTTTTCTGAGACAGGGTTTCTCTGTGTGGCCCTGGCAGTCCTGGAGCTTACTCTGTAGACCAGGCTGGCCTTGAACTCAGAAATCCGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAGGTGTGTGCCACCACTGCCCAACTAAAACCCTAAAGTTTTTGAGAATGGCTGAAAATGATCAGCTAAACCCAGATAGGGAAAACAAGACTGAATTGAATTCAGCCTGTCCAGTGGTGCTGACCGAGCCAGATCAcaatgcccccctcctttcctcacccccagctgtcggtggtcttatggggtctgccttaggaaagaccgtgcacatacctgcattctctttctggccactccttgggcctggggcattgtttgcaagggaagggtggtaaaaggtgtttctatatgaatggacctcctctttgtaggcattctgtgagtgtcagcttggcccagctctcagccaccatttttcgaaaggaaggcctacaaggatggcctcagttcatgaaccttcttcagcacagcactcatagttcccatagccccgagaaagaggtactggcactgccctgttggcaggaaggagggacagaggctggatagaccacgagaccgtctcttctcccctgctcctgtacaggtgggacttctgctgctgagtgtggtggtgtcttcccagcccgaggcattccatgcccaccagcacgagctgcttcagcttctgaatgagacgctcagtgacgtcagctttcccggagtgctcttctactccctgcgcactctgactgccatcgcccgctatgtcaggccggatgatgtggtaagtaagacgctgcctgtatctctcggtttcgtgccctcgctttcagaagtcttggcccaagctgctttcgagttttattccgcctcccgttcttcctaagggttgagggcatagcgtgttagctgggcctggctggctccttagatggaggctttgaggaagcctccagctcctggcttctgggatgagaccacctgcgctttatgtgattacgctcattcctgtgtcttccagtctcttgcacggatgttggtgcccaaagtcgttacggcactgaggactctgatccccttagatgaggtaaggacagacgggaggggtctccttttggaaggaaaatggaggcacaagcaccatggtcccctagaggagcgagtaccctgtgacttacttgtctgtctgcaggtaaaggcctgtgaggccctggaggccctggatgaaatgctggagacagagttgcccatcatcaacccccacctctctgaggtcctcacgttttgcctggaggtgagcccggggtgggtgctgctcctgctcgctggtttgccgttaagttttcttgcttgtttctgattcttgtcggtgtctgtataggtggctaagaatgtggcccttggcgaaccactccgcgtacgtgttctctgctgcctcactttcttggtcaaagtcaaaagtaaggtacgttccctctggggaagttaccccatcctgctgtctccgccattagttgtgtttgtaggcttagtcctgctcgagccctggcccagccttctctccctgcctaggagacggggagtctctggctggggtccttgtggttgggaggctgggattttcccatgagcctgttctgggaagatgctcaaggctagccctcccctcttaggccttactaaagaaccgcctcgtgcctcccttgctgcacgcccttttccccctcatggctgctgagccacccatgggccaactggatcctgaagatcaagattcggatgacgatgacttggagatcgggctgatgggggagactcccaagcactttgctgtacaggtgatgtattggccttctcgtcactgtcacccaatactcagagaaacagacaccttaaggggaggaagtattttcatggcttcggagatttccatttactgtggtgggaaaggaatagccaaacagtggttcaggcctcggcaaacCAGAACCATTCACGTTATGGATAGAAGGCAGAGAAGGGGTATACAAGGAGGAGACAGGGCAAGATAGCTCCCATAGGATGTGGCCCCAACTAAGCTCTGCCTTCTGCCTTTGGCTCCTCCTAGTGACTGGCATTAGATTAAGAGATTAACTCATCTTGAGGTCAGAGCTCTCCTGATCTAATTGGCTCTGAAAATATCATCACAGTTTGTCTCCTAAGCACTTCCCAGTTCAGTCAAGTGGATTATTAAAATCAGCCATCGCACCCAGCTGACCTGGTGGACACCTGTAAGCTCAGTCCCCAGCCCTTGGGAGGCTGAGGTAGAACTGTAAGCTTTGGGCAGTCTGAGCCAT

This mutation is a 1170 bp deletion beginning at Chromosome 14 position 55,632,815 bp and ending after 55,634,584 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48949 TGT ATA CCC CTT CTC TGC CTT C Common A
48950 GTG GTG GGA AAG GAA TAG CC Wild type Forward A
48951 CCC AGA TAG GGA AAA CAA GAC Mutant Forward A
48952 Fluorophore-1 TTC AGG CCT CGG CAA AC Quencher-1 WT Probe
48953 Fluorophore-2 TGA CCG AGC CAG ATC ACA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48949 0.40 uM
48950 0.40 uM
48951 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.