Protocol 35043: Probe Assay - Adi1<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

>chr12:28677478+28677587 110bp GACCCAGCAGGACTCTAGCA TGCTTATTCTTTGCCCATCC

Mutant=  110 bp

Wild Type = 110 bp

Sequence

AGAGGATTGCCACTCCAGTCCATCTCAATGAACTTGAGCTTGGTCTGTGGGCCTGGGAAGCATGCTGACCACAGAGAGTCTAGTGAGACTTGCTGCATGGAAGATGGCAGAGTGGCCACACACACTGAGTGAGTGTTGTCTTGGCATCTGTGAGGAAGAAGGACTTCACCTGCAAATGTGGAGGACCCAGCAGGACTCTAGCAGAACTGGAATTTCcacctggcactgtgaaaatggatgatccatcaagagaggaggtggagggctgtgctaggatgggcaaagaataagcagtgctctgttaggatgatccagccacaggtctggaaggacgtgttgctatgtttccatcacagggccggggagcagacctagagccttgtgcaccactaggcaagcactataccagggagttatactccccgcttagctgctatgcatccttgtgctaaggtgacctccactttgcttaaacttacatagctagatgctgacaagtatgagaacgatccagaactggaaaagatccggaaaatgagaaactactcctggatggacatcatcaccatatgcaaagatacacttcccaattacgaggagaaggtgagcaggccctgctgggagggaatccttaccttcttgcctgctaacacaagacttttccgtgtggtttggggcagagtgatgctttgtaattgtcttagttaatgaggcatactgctgaatgatactcactctttctagttctttctgaactctggctggttcaacccagctgttctggctcaaaaattcttttcaagctgactaattcaaactggcttctcttagctcctgactgaaaaaatctctgcgtggtctcaaactaactctggcaatctgttctaattttctgcaaccaaactatgtaaaactgcctcctccagaatcaccatgcactgctcgcgctcgctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctctttcccgagacagggtttctctgtatagccctggctgtcctggaactcactttgtagaccaggctggcctggaactcagaaatccgcctgcctctgcctcccaagtgctgggattaaaggcatgtgctgccaccgcctggctatgcactgctctcttaaagttacctctttgctgttctagtgagagttgggcgtaccctatctctgactcattctgtgaaatctctctctgattcatcactttgtctgcctctcagacgccattttcaaacatggttgcttccttctgtaaactaaccctcctttcattgtctgggattaaaggtgtggactaagggcatgtttgtatctcaggcagagggattaaaggtgtatgatgtgtctgcgttccagctggatcacacaagcctaggtcttggatgtgatcccttgccagagcacggatcatgttgctgggttaaaattcctttacaatgctgttctcatagaatgtgctggggtgattccctttctttcagctcttttgaggggtatgaatctggtggtactgtgtcccttatgtgtggtagatccaccagtgatgcttggattggcttggggttttcttcatgggattcacagctctgcatctggctctgcccttgacttgttgtcggttggacgatacatttttctgtgagcatctgtgttgtcttcggggtgggtacctgaccacttcactgggaccatttgagcttagtggcatagttatttagagtggagcatggtctcaggtaggtaatctcagcttctggtaggtgaagacaggaggagcagaagttcaaggtcatctcagctacttagcaagtttgaggacagcctgtgctacatgagaccttgcctcagtttgcccctaccacttgggttctgtggattaaatttaagacatcatctcggtggcaggtgcctttaccgacagtgtcatttcctggcttggctcttgacacttttaataccaagaattattcactgtagtctagctgtaagggctgggtctgtgtgatcggtctccaggaaactcacaggttcccccttgcactagatcaagatgttctttgaggaacatctgcatctggatgaggagatccgctacatcctggagggtagtgggtacttcgatgtcagggacaaggaggacaagtggatccggatttccatggagaagggggacatgattactcttcctgccggcatctatcaccgcttcacactggacgagaaggtgagagtgtaccatggtgtggtctgtgcatagacatcgcaggtgaccatgatgacccactaggtgtgttgtgtgaggctcatcgcctagatacatggccattcacagctctttcagagaaagccagaagagcttgtggaaccagaagagcctgtggccatggaacctgggagggaagggtcagaatgtcgcctttgttcttgttggacagcgaggcagcattatctacatggctttggtcaaggggcaggtatcttcaggagggagagctcctgggggggacctgcacttgtcactgACTGGGGCTTGTGGTGTAGTGTGTGGGTGCTCTCATCTGAGGAGCTTCTCAGCAGTTTTGCAGTACATGCTTCAAGGTCACCCGCAGCTATATATCAAGTTTCAAGAATAGGATGGGCTAGATGAGACCCTGTATCAAAAAAACAAACAAAAACCCCAAACAAGCAAACAAACAGATAGTTACAAATGATGAGATGGGTACAGGGTGTTTACCAAT

A 2357 bp deletion beginning at Chromosome 12 position 28,677,511 bp and ending after 28,679,867 bp (GRCm38/mm10). In addition there is a 20 bp AGATACTTATGCTGCCTAGC insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45107 GAC CCA GCA GGA CTC TAG CA Common A
45108 TGC TTA TTC TTT GCC CAT CC Wild type Reverse A
45109 CCT TGA AGC ATG TAC TGC AAA ACT Mutant Reverse A
45110 Fluorophore-1 CTG TGA AAA TGG ATG ATC CAT C Quencher-1 WT Probe
45111 Fluorophore-2 TGT AGT GTG TGG GTG CTC TCA T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45107 0.40 uM
45108 0.40 uM
45109 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.