Protocol 37522: Probe Assay - Pcdha9<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

>chr18:36997754+36997904 151bp CATTTAGCCACTGGATGTCG CTTCTGGTCGATTTCCTAATCTC

Mut = 159 bp

Wt = 151 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

ACATTTAGCCACTGGATGTCGCTGCCGTCCATGAAATGTATTGCTTTTAAGAACAAAGATATAgttcgctgtttctcaggggataggaagtagctacgttcggagatctcggcgtttcggttaaaaaatgagattaggaaatcgaccagaagatattagaacttgtgtgcatctcagatggcacattcatggacttttaagacaagagaatgcttcagtggtgatctccaaatgtctgaggcatggagcttggcgcctgctactttggcttctgctccttgcaacatgggatgtagggagcggccagcttcactactctgtcccagaggaggccaaacacggcaccttcgtgggccgcatcgcgcaggacttggggctggagctggctgaactggtgccccgcctgttcagggtggtgtccaaggaccgcggggaccttctggaggtaaatcttcagaatggcattttgtttgtgaattctcggatcgaccgggaggagctgtgcgggcagaacgcggagtgtagcatccacctggaggtgatcgtggacaggccgctgcaggttttccacgtggaggtggaggtgagggacattaatgacaaccctccgatattctccgtagcggaacaaaagatcttggtagctgaatctagactgcttgattctcgatttccactagaaggcgcctctgatgcagatgttggagagaactctatgcttacttacaaactcagttcaaatgagttttttatccttgatattgtaaacaaaagaggcaaaggcaaatttccagtgcttgttcttcgaaaactgatagatcgagaagaaaatcctcagctaaagttgctactgacagcaaccgatggaggcaaacctgaatttacgggatcggtttctctgttgatccaggtgttggatgttaatgacaatgctcctgtgtttgacagatctgtttacgaagttaagatgtatgagaatcaagagaataaaaccttagtaatatggctgaatgctactgactcagatgaaggaataaacaaagaagtggagtattcatttagctctttagcctcatcgattataagacagaaatttctaataaacgaaaagactggagagataaaaataaacggcgctattgactttgaggagagtaacaattatgaaattcacgtggatgctacagataaaggatacccacctatggttgctcactgcaccgtactcgtggaaatcttggatgaaaatgataacgcaccggagatagtccttacttctttgtctttgccggtgaaagaagatgctcctttgggctctgtaatcgccctgatcagcgtgtcagacaaagactctggtgtcaatggacaggtgacctgctccttgactaaccatgtccccttcaagttggtgtccaccttcaagaattactattccctcgtgctggacagcgccctggaccgagagaccacagctgactataaggtggtggtgacagcccgcgacgggggctcgccctctctgtgggccacggccagcgtgtccgtggaggtggctgacgtgaacgacaatgctcctgcattcgcacatcccgaatacacggtgttcgtgaaggagaacaacccgcctggagtgcacatcttcacagtattggcggtggatgctgacgcacaggagaacgcgctggtgtcctactcgctggtggagcggagggtgggcgagcgcttgctgtcgagctatgtgtctgtgcacgcggagagcggcaaggtgttcgcgctgcagcctctggaccatgaggagctggagctgctgcagttccaggtgagcgcgcgggatgctggtgtgcctgccctgggcagcaatgtgactctgcaggtgtttgtgcaggatgagaatgacaatccgcccacgctgctcggacatcagtcaggtggacctgctggcgaatttagccagcttgtttcgcggtcagtgggtgcgggacacgtggtgtcaaaggtgcgcgcggtggatgcggactccggttacaacgcgtggctgtcttatgagctccacccaactgtgggcgctcgcagccctttccgtgtcggtctctacacgggtgagatcagcatgacccgagccctggatgaatctgatttaccgcgccagcgcctattggtgttggtgaaggaccacggcgaacctatgttgatcgccactgccaccgttttggtgtccctggtggagaacggccaggtgccaaaagcttcttcccaggggttgcccaactcctcccgcagggaggcgtcgctgatggatgtcaatgtatacctgatcattgccatttgcgcagtgtccagcctgttggtgctcacgctgctgctatacacagccctgcgctgctcagctgtgcccatgcaggctgggtgcgggcttgggaagcccactctggtgtgctcaagcgctgtggggacctggtcatactcacagcagagacagcagcgagtatgctcaggagagggtccgcccaaaaccgacctcatggcttttagccccagtcttacaccttgcccagtggccgaagtgggaatggaaagtcattctgttggaggagacgtgcccggaaaggtgggctattactttttactctcatcttgcttgatgaaatacattttctgtctcattttattttatgacaaatgtgtgtcccagatgtatttttttttgtctgagtaccactatgcacaaGTATTTTTAACAATGCGTTGGAATATAGTTTATTCCAGCAATTGGTTTTAAGATGTCCATCTA

This mutation is a 2665 bp deletion beginning at Chromosome 18 position 36,997,816 bp and ending after 37,000,480 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51580 CAT TTA GCC ACT GGA TGT CG Common A
51581 CTT CTG GTC GAT TTC CTA ATC TC Wild type Reverse A
51582 ACA AGT TTT AGG CAG AGG TTT G Mutant Reverse A
51583 Fluorophore-1 CGC TGT TTC TCA GGG GAT AGG Quencher-1 WT Probe
51584 Fluorophore-2 CGT TGG AAT ATA GTT TAT TCC AGC AAT TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51580 0.40 uM
51581 0.40 uM
51582 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.