Protocol 46398: Probe Assay - Dpy19l4<em1(IMPC)J> Alt1
Version 2.0

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=  123 bp

Wild Type = 122 bp

chr4:11303593+11303714 122bp ACAGAGACCCTCGATTCTGC TTATGGTTGGGATCAGCACA

Sequence

Wt Sequence (deletions in lower case and insertions with carrots ^g^ (g insertion):

CCTCCTGCCTCAGCCTCCTCTGTGCTAGGGTTGTGGGCCTATACCACCATGCCTGGCTTTTACTTTAGAGTTTGACATTCAGTTTATTGCCAGTCCCTTCTTACTGTTCctgct^aa^GTTTGTAGGGTATTCTTTGGGTTTTCCTTCTAGAATTTCCTTGTATGTAAAACATATAATGttatatcagttgtaaaccatttgaatgttttaacaaaaatgattgctaggctattgagaaatttaatcttattaatacagtagtttaggaaattttaaatatgtccttgaatacaactgtaaatgtcagtttaaaatgtgaaggaaacaaacgaaaccaaacacaagaggctggaggagaagaaagagtgcagcctgacctttactgcttgtgtcactgcaggagctcgagcgagagatcaccttccagggagacagcgctatttactactcctactataaagacatgctgaaggcgccgtcattcgagagaggtatggccagccacagcggtaactgcagcaatgatgttgttctaaaggaaagcactcttctcaaaggttatgagctttaggatgcgggatatgatagtaagttgggaagatgcagcatctactgtaacatctgagtgcctttttttccatattatttgtgattactaatgaatgacatgttgaatcactgtcggggcagaacagtaatctggagaaaaatcatgtatttaggtgtttatgagctgacgcacaataacaaaaccatctccctgaagactatcaacgccatgcaacagatgtctttgtacccagaacttattgccagtgttctctatcaagcaactggcagcaatgtatgtatttattattattattttttttattttagatttacttcatgtgtatgagtgttttgcttgcatatctatatgtgactgcatgtgtgcccagtgcccttagaggacagagagcatcagatcctctaggacactagttctcactctgtgatattctgcatatcaggtgtttacattatgattcaaaacagtagcaaaatgacagttatgaagtagcaatgaagtaagtttatggttgggatcagcacagcatgagggactgtcacagcactaggaaggctggaagccactgctccagaagttgtagacagttctgagccactgtgggggagcagaatcgagggtctctgtaagaagaaaagtgctcttagccacggagccctccctccagccctgtgtcttcactgtgacgtctgttttcctacgtgggtttgtatctttgggttgtttaaccatcctctgtcactccctttctttttactttaggaagtgatcgaaccagtgtatttctacattggcattgtgtttggattacaaggaatgtatgtcacggctttgtttgttacaagttggcttatgagtggcacgtggctagcagggatgcttactgttgcgtggttcctaattaacaggtatgcaaggtttttacattgatatttacagattttgttttggttatagagttccaaaaatatccccattcttacagtgctcagctgttatctgcaaacctattgattgttatgtttgatggtgacgattaggtaggactccacacagggactaacttagatacaggatcacttacagagccggtctaagcgcttgCTGTACAAGTATGTGAGCTGGAGTTCAGACTCCAAGAATCCACCCTTCTCAATAGTCACAAATGATATAAAATACTTTGGCGTGACTCTAACTAAGGAAGTGAAAGATCTGTATGATAAGAACTTCAAGTCTCTGAAAAAAGA

This mutation is a 1497 bp deletion beginning at Chromosome 4 position 11,303,116 bp and ending after 11,304,612 bp (GRCm38/mm10). 61 bp before the deletion site there is an indel with 2 bp insertion (AA) and 5 bp deletion (CTGCT).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66475 GGG TGG ATT CTT GGA GTC TG Mutant Forward A
66476 Fluorophore-1 TGC TGT ACA AGT ATG TGA GCT GGA GT Quencher-1 MUT Probe
67927 CCC TTC TTA CTG TTC AAG TTT GTA G Mutant Reverse A
67928 TTA TGG TTG GGA TCA GCA CA Wild type Reverse A
67929 ACA GAG ACC CTC GAT TCT GC Wild type Forward A
67930 Fluorophore-2 CTG TCA CAG CAC TAG GAA GGC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66475 0.40 uM
67927 0.40 uM
67928 0.40 uM
67929 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.