Protocol 32805: Probe Assay - Ier3ip1<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

>chr18:76941148+76941268 121bp TTGACCTGAACAGTGAGAGCA TATGGGCTGATTTTGCCAAC

Mut= 125 bp

Wt= 121 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AATGCACTTTAAAATTTGCCCACTCAGGCGTTTTCATACTGATACTTGGTTAGATGACAGAACTCTTAGACTGAATTCGTAGTTACTCACCCTAGCACTGCATTTGTTTTTTAGTTCTTCATTGACAAAAGATTCTTTTGATAATTTTGAAAAAGGAAAAAAAAAATCAGGTTGTGACAGTTGCcatctttactgaactgaacaccatgaaatcatttgaactgtagattttgtaaaatcatagtgaaatgaagaaaatatggttgttgccactatcaaaagaaaaaaattaaagaactcaatattttaactttaagatgtatgagtttatacttaatatcttcacagggctcaaactagtttttaaaagatggctaatgagggaacaagacaagtgcataatacatttaactcaactaatcatctttgtcttgcacagttggctggggaacagaccagggaatcggtggatttggagaggagccaggaattaaatctcaactaatgaaccttattcggtctgtaagaaccgtgatgagaggtaagaagaacttagaggacgttataagttacagtccgtggtccagtttcagaccccaaagcgtgggtgaatgtgagctctcaggttctctttgtgtggaacatggacttctgtaacctgaatttactgcttcagagttgctggccgctttcttccattgcatctgacttctaggaagttcagtgttactagtacactctgtgtgtttcctgtcagggcagaaagacaaaatgatgtccttactgcttaggaactgtagctgccatcttgtaatgtagagtcatgaagaagtttccaagagtgaaataaaggctctgtattgtattgaaaaatactttggagcaactcttcaatcaagaaagagttcagataccttgtatttaaacaatgggattagagccttttcaaaccttaatcatagctttgggtttattgttaataattttattctttttgatgaaactgcagaaccttccttgaactgtatttctgctccagtgtgcagctgtgtggatctgtctgatcctttgtgttcccagagctgcagcagcaccatccagtggtgcctccaaacacccagacatatggtggggtggggcatggtggtccacacctgcagtcccatcccttgggagggctacagaggcattcctggttcacctccatccaacaaaagaaaatgtctttggttttatggtcatttatttttctgttattactattactatatgattatgatggcatatttatagatacttccatactcctcggaagctatttattgctgtttaattttttgtttgtttgttttttatactaacaaactttatttttttcttttagtgccattgataatagtaaactcaattactattgtattgcttctgttatttggctgaacatcgttgggaaatcagagactgagaagaagaaaagccccagaagttactttctgttgtgaccggaatattcatgtttcagcaggctcatcttgcagttgacaaaaaaaaaaagaaaaaaaaagtaaagttgacaataaaaccagagtttctatctatctgatttttttttttttttaattgtagttgcacttgtagatcagattatcagaggcaggagcactcccggattggagcatacttgttttagctaaaataggtaaaggctaggaaagggcacaaaactgctgcctttgtgtcactccatacgacaccctgttacagctcagggatggatttagatataatctgagagtatataacttcctatataaacttaagagtttttttaaagtaacagttacatctagttgacaaattggtatttgggtaatttattttgaactctttggttacctcgtttctgctataaagaaatttgtaggtaactctacggggcaatgccgtgatttcagagagaattctaattctggaaaccagttgtttgggaactgtggtttttacgatattatttattagttgacctgaacagtgagagcagactgcttacatattttcctttgtaggaagtcttgcctgttttacaaggatttgttttataaaaatcagtgtaactgttagttggcaaaatcagcccatacagtcagactagcctgctcccgtgactgtctgttaggaagcctttgccttccttaggttgtgcttccattcctcggcggagtggcatgctgtaggtatgattcttaattctgtctgtaggtaggttgttgattctaagtagctttcgtttgttcgtctgtcttatgtcttagaccagctttgtggtgttcttggctcatctcataatactgttgttcagtttatatcagaagagttaaactgtgcaaatattgtatgaaatgtttcagttaccataatttaaaggggtgggatggcatctttctgtatagaaaacttttataaataaagttgacttttcatattaagcgtctgatttatgcctttatatagacctggcattatgagtttgttgatccctcttttattcttcactgtaaaattgctgagtagaaatttaatgaatgttaatattcataatctatttgtttcttcagtgaattaactgaattgttgacataaatcaagagaatccttgcgtaacttttaagttactgctaactggggcaggtattgaagatctcctggtaatgtctcaattggagagagtgggtctttccttcagtcttcagggctgtcagcggtacCAAAAAAGTGTCCTTGAATCCTTGGCCCCTCCCCTGTGAGGAGCTTGGTCCCAGGATGTTCCCTTGGCCTCAAAGCTAACAGGCCAATCTTGCTGACTCACACCTCATTTTATTTTATTTTTTTTAATTGTTGAAGATGATTGCATCCAAGTTTGAGCCTCAAATCAGATCTTTAAAA

This mutation is a 2581 bp deletion beginning at Chromosome 18 position 76,939,313 bp and ending after 76,941,893 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40040 TTG ACC TGA ACA GTG AGA GCA Wild type Forward A
40041 TAT GGG CTG ATT TTG CCA AC Wild type Reverse A
40042 TCA CCC TAG CAC TGC ATT TG Mutant Forward A
40043 GGC CAA GGA TTC AAG GAC AC Mutant Reverse A
40044 Fluorophore-1 TTT GTA GGA AGT CTT GCC TGT TTT A Quencher-1 WT Probe
40045 Fluorophore-2 AGG TTG TGA CAG TTG CCA AAA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40040 0.40 uM
40041 0.40 uM
40042 0.40 uM
40043 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.