Protocol 45819: Probe Assay - Ankrd34a<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

Mutant=  117 bp

Wild Type = 119 bp

Fam=Mut

Hex=Wt

chr3:96597447+96597565 119bp CCGGCTCCATCGGTGTTT TCCTCCCTCCAGAAGCAA

Sequence

Wt Sequence (deletions in lower case):

TAGGCTTCACTACCGAGACTTCAGGCCTCTCCCAACCACCACCCCCATACTCCTAGATATCTTCGGCAGGTCCCAGTCCCGGCTCCATCGGTGTTTTTGCCCGAGCCGCAGCTATGCTGCACACCGAgggccacgctcttcttcgagctgtgggacagggtaagctacgcttggcccgtttgcttctggagggaggagcctacgtgaatgagggtgatgcccagggggagactgcgctaatggcggcctgtagggcccgttacgacgaccctcagaacaaggcgcgcatggtacgctatctcttggagcaaggcgcggatcccaacatcgcagaccgtttaggacgtaccgcgctgatgcatgcttgcgccgggggtgggggagccgcagtggcctctctgctccttgcccacggcgcagacccttcagttcgagatcacgctggcgcctcggcgcttgtccacgccctggaccgaggggaccgcgagacccttgccacactgctggacgcctgcaaggccaagggcacggaggtcatcatcataacgacggatacctcgccctcgggcaccaagaagaccaggcagtatcttaattccccaccgtccccaggggtagaggaccctgcacccgctcctcctagtccaggggtctgcacatcgccttcagaagtccaactgcagactgcaggaggagggcgcgggttgttatcccctcgcgcccaggaagaagaggagaaaagagacatttttgaatttcctcttcccaagccccctgatgacccttccccttccgagcctctccccaagccacctcgacatcctccaaaaccactcaaaaggctcaattccgagccctggggccttgtggctcctcctcaaccggttcctcctgcagaagggaggcccggtttggagcgtctggctgctgaattcaatggcttgaccctgaccgggagaccgcgtctttctcggcgtcacagcaccgaaggcccggaggacccgcccccatgggcggagaaagtgacgggtgggggtcctctctcgcgccgaaacactgcgcccgaggctcaggagtctggtcttccttcagggctaaggcaaaaactgagccgcatggagccggtggagctcgacacccctggacatttttgccctgactcccccgagtccagccggttatccctggagcgccggcgatacagcgcctctccgctgaccctccctccagcgggctcagtttcctccccacgccagtcccaggaaagtctgcccggggctgtatccccgctgagcggacggaggcggagtccagggctgctagagagaaggggctccgggaccttgctcctggaccacatctcgcaaacacgaccaggtttcttgcctcctctcaacgtcagtccccaccctcccatccccgacattcgtccccaacccggaggtcgggcaccctcactgcctgcccctccaccctctggggcaccagggtctcccaggaccaagcgcaagttggtgagacgccactccatgcagactgagcagattcgcctgctaggggggtttcagagtctaggtgggcctgGGGAACCCGGGCGCTGAAGGAAATGAGGGGCGCCAAGGAGGGGGAGGGTCAGTACCCTGATAGAGGGAGAACCAGCTGATACCTCGGACACGGGATCTCTTGCATGTGCTC

 

This mutation is a 1457 bp deletion beginning at Chromosome 3 position 96,597,496 bp and ending after 96,598,952 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66521 TCC TCC CTC CAG AAG CAA Wild type Reverse A
66522 CCG GCT CCA TCG GTG TTT Common A
66523 TCC CTC TAT CAG GGT ACT GAC C Mutant Reverse A
66524 Fluorophore-1 CTG CAC ACC GAG GGA ACC C Quencher-1 MUT Probe
66525 Fluorophore-2 TCG AAG AAG AGC GTG GCC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66521 0.40 uM
66522 0.40 uM
66523 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.