Protocol 34881: Probe Assay - Iqch<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

>chr9:63525247-63525384 138bp CGTATGTAGAATCAGTCCTAGCAG CCTGAGGAAAGAAAGTTTTCCAGA

Mut= 129 bp

Wt= 138 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ATATATTTATTATATATAGAGAGAAGTACTTTTAGAGCAGTTTAATAGCTTCTCATGAGATATCCCCCAGTAATGAGTTTTAGTGACATTTCTTTGACATCCTTATGTACGTATGTAGAATCAGTCCTAGCAGCCATGCcttcccatcactgggtggtaattgatgaagtgttagtaggtatgtactgatcacactcttacagagatcatggaatattttaattctggaaaactttctttcctcagggcatgaagattaggacaccgtctaagactaggaaatcaccatgggattatatattttctgtttacaacggctgtgtagacaggacagccccagacttcttagcattcaaatcacgcttcaaattaatctgggggagtattttttctttcctggaacaaattgagaaatttctcaaggactatgcaatatctgaagccaaaataaaagggaagagtctggtgtccctgcttccagagtttgagctgaagaacaaattaaccagaaatgatgttctggctgtgttagagaacccagttcacattcagatgctgttaaaccttccgggacagaggtacaagggccaagatggaaaggcagaagctgccaccaagatccaagccacgtggaaaagttacaaggcaagatcttctttcatttcttatcgccagaagaaatgggcctcaggtgtgatcgccctggcgtggcttttacattgccacaggacaagactaaagaggatagtgaaggaatcccgacagagacacctggagaacttccgcattcgagcccaggtatgccaggctgccagctgttaaggcaagcctctttttctaaatatttcctgcttgaacaagtgcagtaacttgtctctttatctttccttccctctccagcctccctccccctcagcattctgcatctataattctcctcaggatatgatacacaaactcagagcctggcctgcatgcctgcaaaatctgttataatttttctataaaagaaaatggtccttgttttgactatggtagttgtgttgggaagtcactgttctaactgcattaccttttctatacacacacacaacacacaagcgagcacgagcaaacacacacacacacacacacacacacacacacacacacaccttctgacactttaatttggtaggaaaaaacatcctcacttgtctatgttgctgtcaccactttgtcagaaagtatttaagtttgcagacagcCTTTTAATTAGTGCTCACAATTTCATTTATGTTTGCTTTCTCTTTTTTTCATTCATATTCTGTTAATTAAGTGAGCTGCCTCTAATCTTCCCCTGCCAGTAGCATCATGTAGCCACCTAATTAAATTAATTGGGGAAGATGTTTTAAGTAT

This mutation is a 1108 bp deletion beginning at Chromosome 9 position 63,524,247 bp and ending after 63,525,354 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44689 CGT ATG TAG AAT CAG TCC TAG CAG Common A
44690 CCT GAG GAA AGA AAG TTT TCC AGA Wild type Reverse A
44691 CTG GCA GGG GAA GAT TAG AG Mutant Reverse A
44692 Fluorophore-1 CCA TCA CTG GGT GGT AAT TGA Quencher-1 WT Probe
44693 Fluorophore-2 AGT GCT CAC AAT TTC ATT TAT GTT TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44689 0.40 uM
44690 0.40 uM
44691 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.