Protocol 35228: Probe Assay - Ces5a<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

Mut= 158 bp

Wt= 166 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAGGCAGAGATAGCTTGTGCAAAGCCCGGGGGCAGGAGCCTTCCTGTATCCGTCTGCGTTCCTGGTATGGAGGCTGTCGAAAGATTCCTTTGTTACTCTGGCTGTAGAAGAGAGACTGGTGGGAGAGAGTCTCTGCAAAGCAACTCAGTGGTGGGCACAGACAGggcgtgtgcctggggttaatgacataactattttggaaaatgagctttctatccaggaaaggggctccccatatgttagtttgcttgcttgtttctgaaggaaagagcccggggcacaatcagattgtgtgccaatcgttttgacatgcctcttgctcagaagggatgcctggccttgcttctgtcccaggaaggcatgctgacagatgcggagagagtaattctcccaccccatctctttcccctctccctcatcctctccctctccctccctcacaggtgctttcagaacttagagtggttattcatatatcaaaacctgctcaaagtaagttaccctatacttggaatgtcagaggactgcttatacctcaacatttatgcaccatgccatgccaataacggctccagtctcccggtaaggctgcctgctgccctggggttcattagcaggtatgggtaacagctgggcatggggcatctggagttagacctagcctcccattgggtaagaggaaaagtccatcacacaagtaacttgtttgttggttggttttttttcttttctttccctcgagacagggtttctctgtatagctctggctgtcctggaactcactttgtagagcagGCTGGCCTCGAACTCAGAAATCTGCCTGCCTCTGCCTCCCAAGTGCTGGGATTAAAGGTGTGCACCACCATTGCCAGGCACACAAGTAACTTTTAAAAACATATTATAATGGAAGCTGTCCTCTGTCATTGTCTCTGTTGAATATTTCG

This mutation is a 631 bp deletion beginning at Chromosome 8 position 93,530,546 bp and ending after 93,531,176 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45562 AAC TCA GTG GTG GGC ACA GA Common A
45563 CAA AAC GAT TGG CAC ACA Wild type Reverse A
45564 GAG ACA ATG ACA GAG GAC AGC Mutant Reverse A
45565 Fluorophore-1 AGG AAA GAG CCC GGG G Quencher-1 WT Probe
45566 Fluorophore-2 CAC CAT TGC CAG GCA CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45562 0.40 uM
45563 0.40 uM
45564 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.