Protocol 35273: Probe Assay - Ces3b<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

>chr8:105086275+105086382 108bp TTTGAACAAAGGGTGTTGTCTC TAGCAGGACAGCCAGTCAGA

Mutant=  109 bp

Wild Type = 108 bp

Sequence

AACCAAACATGAATGTTGAATTTGAACAAAGGGTGTTGTCTCTAACTGGGGCTAGAgtcctagcagggaatgaggacaaagagccatcttcaccatctaagagccagctctgactggctgtcctgctacatctacccacaacacatactaggaaaacaggtgattcacgggcaagcaaggactgggaagaggagaagtgcccagtgccctggtgactgactcaaagcaaatgcacagagtgtaagaaaaaaaatatgtcttggacgctatcttaagccaatcagtgccactccctgtccctagcactggagacaagcacatgccaggcaacaggggattcctggatgtggtggctgctcttcgctgggtccaggggaacatagccccctttgggggtgatcccaactgtgtcactatcttcggtaactctgctggaggcatgattgtctcatccctggtgagtgaccacagagaactagcagtcgaggttatatctgggaaggcttctatggcctcagacctccatgctcctctgcctcctaacatctgcccctctgttgttcccagttcctgtctccaatatctgctgggctcttccacagagccatatcccagagtgggattgtcacgaccattatgatggaagacatgaaaccatggcctgaagctcaggtctgcctttccttcactctcttaccctaagtgtgcctccacagcccaccctgtgctaacctttgggtcaggtgtggagaggaaggagtaaccactagggagtcactgataggctcagagacaattggctagtgcagtccaggaggtaggcagtcagacaaggctttctagaggaagccaccctggaagaggttatctctggttcttctctggggttatttttgtagatcccccccattccccaacgGATTCCTATATTTCCAGCTACTCTGTTACCTAAGAGTTCGGTCCCCTTTTGAGGAGTAGCCAGAAGTCACCAAGCAATCCCTAATAGGAACTGTGAGTCAAAAGCTTCATGCATAATAAGACAGGGATGGGGCACAGAAGCAGTAAAGCCATCAGGTAGGGAACAAGCAGGAAGTGAATGAACTAAAGGAGTGGAAGGTAGTGCAGGGAGCTGACAGACCATGCCTGGGAGGGAGTTTGGCGGGATGCAAAGGGCTAGAAGAAGCAG

An 863 bp deletion beginning at Chromosome 8 position 105,086,311 bp and ending after 105,087,173 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45670 TTG AAC AAA GGG TGT TGT CTC Common A
45671 TAG CAG GAC AGC CAG TCA GA Wild type Reverse A
45672 TTG GTG ACT TCT GGC TAC TCC Mutant Reverse A
45673 Fluorophore-1 TGA GGA CAA AGA GCC ATC TTC Quencher-1 WT Probe
45674 Fluorophore-2 CTG GGG CTA GAG ATT CCT ATA TTT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45670 0.40 uM
45671 0.40 uM
45672 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.