Protocol 42761: Probe Assay - Ankrd13b<em1(IMPC)J>-Alt1
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

Mutant=  135 bp

Wild Type = 130 bp

chr11:77476814-77476943 130bp GGTACTAGAGACAGCGTGAGCA GGCGGTACAGCTAGAAAAGAAC

The wt reverse primer (primer 59965) anneals over the nucleotide sequence containing mouse genomic variations rs214155141 and rs240652107. 

Sequence

Wt Sequence (deletions in lower case): TCAGTAAATACCTGTTGAAAGCTTGGATGAGGTTGAATGGTGGCATGAACGCACCGTGGGTACTAGAGACAGCGTGAGCAAGCCATCCCCatgcccagcctcagattgtttaaatctccgttaggggtgggggtgaaggaagaacctctcccgagtgagctccctagttcttttctagctgtaccgccagctccaacgcctgtgccccgagctggcccaggaagctgagtcctttctgaacttcccttccacccagtgcctctggtatccaagatttgccctagtgacacatacaaagtgtggaagagtgggcagaacctgagggtagacaccacgcttttgggctttgaccatatgacctggcagagggggaatcgcagcttcgtcttcagaggtcaaggtcagtgaggcagtaagtctggcagggtgggagacagcggacagccccagactgccctgacccctgtgctgttccctggtggcagacacaagtgctgtggtcatggagattgaccatgaccgccgcgtggtgtacatggagaccctggcgctggccgggcaggatcgggagctactgctggctgccgcccagccctcggaggaacaggtgctgagcaggcttactgcgcccgttgtcaccacacagctcgacaccaagaacatctcctttgagaggtgagctggtgtagcctgggtggccaggagccagcccgatgggccattctttcacctggaccactctacctccccaggaacaagactggcattctgggctggcgcagtgagaagacagagatggtgaacgggtacgaggccaaggtgcgcacactcctgatccactttccccagcctgtgggtgcagaggcaacccagcatggaggaaaggcaggagaccaacaggttccccaggagaaggccgtttgcagaatccttagggtagtcacatccaaggctctgttcagacacgctgtatgggtgcagagaaacttgtaccaccctcatttcctccccacaacaggcagatgctcttatccccactctatagatgaggtactgagctttccccacagaccccacttaatcttaagcactccaccaagCTGGGGTTGCCGGTTGTTATCCTGGACTACAGATGAGGCCACGAGGATTGGAGTGGCTCACTGACCTAGTAGAGG

This is a 1010 bp deletion beginning at Chromosome 11 position 77,366,728 bp and ending after 77,367,737 bp (GRCm39/mm39). There is a 4 bp insertion AGGA at the deletion site.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
59964 GGT ACT AGA GAC AGC GTG AGC A Common A
59965 GGC GGT ACA GCT AGA AAA GAA C Wild type Reverse A
59966 GTA CAC ATA GCC TGT AAG TGC TTC Mutant Reverse A
59968 Fluorophore-1 TGA AGG AAG AAC CTC TCC CGA G Quencher-1 WT Probe
61474 Fluorophore-2 CAT CCC CAG GAC TGG GGT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
59964 0.40 uM
59965 0.40 uM
59966 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.