Protocol 34923: Probe Assay - Ascl4<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

>chr10:85927489+85927598 110bp AGGAAAATACTTCGTGCTATGG GTTATTTCTCGGAGTGCTTACTGT

Mut= 97 bp

Wt= 110 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AAGCCTTCAAAGCCAAATCCTTTCTCCTTGGGATTGGGGCTCTCTTCTTTTCAAATGTTGTTTTCCAGTCCTCTGGGGAGGAAATAAACTTAATGATGAGTACAATAGGAAAATACTTCGTGCTATGGGAAGATATTGTTATATCCTGAGTGggattagaagaggttgggaggcaggactaaaggtgtgtttacagtaagcactccgagaaataactggctaaaagtgtttgttactgttcgggcttttgatgtgtttttacagagcagcagaagcacaaagggccccgcgttaaacataccttagaacacaatgtgcggatccttacattcgatgctaaagtacatatattttgttcttgattcctttgaaaagtgtctaatttggtttttaaagattctatttcgcagaagaaaacctcgggcttccccggaggtccaaggaggacttgacacatcgtctaaccacaagagagcagtcttccctttgatgagcgcttcttctccacctccccagaaatgaaagagcagagaggtatgtttgcaaacactctgaaagctctttccatcagacctgaacgccggaaaccacaaagggcagtcaagtttggattttcaaggagaacgccggagtaaactttcgctctgttggccagccgatcagccagtgcctggggaggctctctgctcaattcctgaacaagttcgggctcccggatagccacacttgctgacccaaggagtgaattacaacaattgggcagctttgaaagatcaatctgcttagagtgcgaccgtctatccaggctgggaaccctatgttaactcttaaactaagatcagaggaagcacatggatacaagcgttgatgaggcgagccctctatctttgccccttcttctctgaagctttataaagacccaagaagagcaacctgcttcggaaatcatgcaaagttcactccaagtgtgctcaggtacctaacttctggacctagaaacttctccagagacagaccgggaagtctggatggaccagaatctccgctacgacagcttccagtctcgtcttcaaggtgagagagtcatctgcgtgctttttcgcagagaatggaaaaacgcagatcggccggactgcttgccctgccgtccgcgctacgtacagcgccgctgggcgcgctgcccgggagggagccctgcagggtctcggtgcgccaggatgccgcggactgcgcgcggagacggccgtactcgtccctgccgcttgggggcgtcgccgagcccgcctttttgcgccaacgcaacgagcgcgagcggcagcgcgtgcgctgtgtgaacgagggctacgcgcgcctccgccagcacctgccgcgcgagctggcgggccagcggctcagcaaggtggagacgctgcgcgccgccatcagctacatcaagcaactgcaggagctgctggagcgccaccggccggactgcaacagcgatggcgagtccaaagcatcctccggagcctcgccctgcagcgagtctgaggagcgtagctagcagcatcctagcccactgggctttcatggttgcttctccgggcctagatctttaaaacaaaaccaaaaaccagaaaacaactaagtgcttctggatctaggaaagaaaggtaattgaatgtttagagagtttggttttttttttttttttttttttttgtcccctttcaccttcattggttccgagtttgcagcttaaaaatgcttaaggaaactcaattctcgtatttttgaagcacagaagggccatctcaaatctatgcggtgattttaaaaacacaaaggactttaagaaaccttagccagagaagaaacacacacacacacacacacacacacacacacacacgagacagtccttcacgcgagaggaacattctcactcattccttccaattgggaaatgcactggaaatctcaacacctcactgagggtaggacactccataagacatttgtggagcatctgctggacctgaacgtcgctgtgctaaaaaggacaatgcttgacctatcttgcttcgtccgcaagaggtagatcggaaactttagcagctaataaagtgccgcgaagttaatgatctttttggaggagctctgaatggccatcttctctcccggtttctgcccactgtttctcttgcccttttgtctatgtgttcccctgtacagttctcatcccgtctaacctttgcttggaaaaaataaaaaagcaagtatgcttagtgccttttggtgtttgacactgacttgacaaatggaaagcaaatatgcacggtgccttttgGCATTTGAGCTGAATGGAGAGAGCTATGGACGATGGGGTTGGGTTGAACTTGCTACATGTCCGAGCCATTTGTCTTTGAAAGCTTTCTAGCGTGTTGAATATTTAACTATGGTCATTCCTTCTACCTCACCAAAACAGTTATGAATTCTGTAAAACCACTAGATGCTTGTTGACAT

This mutation is a 2177 bp deletion beginning at Chromosome 10 position 85,927,535 bp and ending after 85,929,711 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44815 AGG AAA ATA CTT CGT GCT ATG G Common A
44816 GTT ATT TCT CGG AGT GCT TAC TGT Wild type Reverse A
44817 AAG TTC AAC CCA ACC CCA TC Mutant Reverse A
44818 Fluorophore-1 AGA GGT TGG GAG GCA GGA Quencher-1 WT Probe
44819 Fluorophore-2 CTG AGT GGC ATT TGA GCT GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44815 0.40 uM
44816 0.40 uM
44817 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.