Protocol 37124: Probe Assay - Pcdhb9<em1(IMPC)J>Alternate 2
Version 3.0

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

 

>chr18:37400953+37401101 149bp TTATGAGCAACACGGTGAAGA CATTACTTGCCTGTTTAGGTGA

 

Mut = 140 bp

Wt = 149 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 TTTGACACTAAAGGGTTGTTTGCCTTAAAGCTAGAATTGTATCAATTATCCCAAAAAGACACAGGGTGGCGCTGCAGGCTAAGATTGTGATTAAAAAATCACTAAAATACCCGGAATCACTGTTGCAATACAGAACAAAGGAAGCAGACAAGAGCTTTGCAAGCACTATGGAGCTATAGAGGGTTTGGGGCAAGAATATAATGGATTAATATGAGATTATGAGCAACACGGTGAAGATTCCTCCAAAgagggaatcagaattcagtgtttcaaaacatttggagactgctacaggattggatgcaagcatgcatttcctcatcatggagaagctggggagaattcacctaaacaggcaagtaatggcatttatttttatgatggttttggttcaggtttgcagtgagccaacaattcgatactctatcttagaggaaacagaaagtggctcctttgtagcccacctggccaaggatctgggtctgggagctagggagctggctgccaggtctgcacgggtgttgtctgatgattacaagcagagattactgctggatcctgagactggagatttgcttctgagggagaaagtagaccgggaggaggtgtgtagcactgtggatccctgtgtgctgcatttccaggtgactcttgaaaagccagtgcaatattttcaaagagaattattgatccaggacataaatgatcatgctccagaattcccagatagagaactgctcttgagaataccagagaacagccagcaaggcacacaattttcactgaatttagcccaggacttggatgtgggcagcaatgggcttcaacaatacacagtcagccccaacccttattttcacgtcctcactcaaaataatagtaaaggcaaaaaatacccagagttggtgcaggacagaggcctggacagagaggagcaggcagagctgagcttaaccctcacagctctggatggcgggtctccaccgaggtcaggaacagccctggttcgaatcctgatcatggatatcaatgacaatgctcctgagtttgtgaacagtccctatgaggtgcaggtcctagagagcagtcctccagactccccagtcttgactgtcttagcacgggatgcagatgctggcaactttgggagagtttcctatggcttctttcaagcatcagatgaaatccaacaaacattctcaataaacgcaactagtggagacatgcgattgaaaaagaaattggattttgaaaaaattaaatcttaccatgtggaaatcgaggccatagatggaggtgggctttctgggaaaggctctgtaacgatagaggtggtggatgtgaatgacaatgccccagagctgaccatatcttcactgaccagctctgtcccagaaaatgctcctgagacgatcataagcatcttcagagttggggacagggattccggagagaatggaaagatggtttgttctattccagagaatctgccatttattctgaaatccaccttcaagaacttctacaccctggtgacagagagccctctggacagagaaagcagagctgagtacaatatcaccatcatggtctctgacatgggcacacccaggctcacaacatggcacaccataaaggtgcaggtgtccgacatcaacgacaacacccccgccttcacccaaacctcctacaccatgtttgtccgcgagaacaacagccctgccctgcacataggcaccatcagtgccacagactcagactcaggctccaatgcccacatcacctactcgctgctgccgccccatgatccggaactggccctctcttcgttaatctcgattaacgctgacaatgggcagctgtttgcactcagggcgctggactatgaggccctgcaggtcttcgagttccacgtgggcgccacagatggaggctcacctccacttagcagccaggctctggtgcgcgtggtggtgctggatgacaatgacaatgcgcccttcgtgctctaccctatgcagaacgcctcggctcccttcactgagctgctgcctagagctgcggagccaggctacctggtcaccaaggtggttgcagtggaccgcgactctggccagaatgcctggctatccttccagttgctcaaggccacggaacctgggctgttcagcgtgtgggcgcacaatggagaggtgcgcaccaccaggttgttgagtgagcgcgatgtgcccaagcacaggctactgctggtggtcaaggacaatggagagcctcagcgctctgccagtgtcactctgcaggtgctgttagtggatggcttctcacaatcttacttacctcttccagaggtagcgcgggatcccgcccatgaagatgaggacgtgcttacattgtacctggtcattgccttggcatctgtgtcttctctcttcctcctgtctgtgctgctgtttgtgggggtaagactgtgcagaagggccagagaggtctctctgggtggctgctctatgcctggtgaacactttcctggccacctggtcgatgtcagtggagcagggaccctgtcccaaagctaccagtatgaggtgtgtctgaggggcgactctgggactggtgagttcaagttcctgaaacccatgatccccaatgctgggatagaaattatggaaagtccccactGCAGGGATAGCTTTGTATTCAACTAAGTAATGAATCTTAAGAATTAATACACTTTTATATGGTTAAGTGTAACATTAACTAACTTTAGGAGAAACTACCTTGTGCATGTCTCCAACCGCTTTTGTTTATCTTATTCATATTACGCACGCTTAGTCT

This mutation is a 2432 bp deletion beginning at Chromosome 18 position 37,400,984 bp and ending after 37,403,415 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48946 GAC ATG CAC AAG GTA GTT TCT CC Mutant Reverse A
48947 Fluorophore-1 ACA TTT GGA GAC TGC TAC AGG ATT Quencher-1 WT Probe
50580 TTA TGA GCA ACA CGG TGA AGA Common A
50581 CAT TAC TTG CCT GTT TAG GTG A Wild type Reverse A
50582 Fluorophore-2 TCC AAA GCA GGG ATA GCT TTG TA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48946 0.40 uM
50580 0.40 uM
50581 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.