Protocol 34486: Probe Assay - Ing5<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

>chr1:93811938+93812038 101bp CCTACGAGATGGTGAGTGCAG ACATTTCCATGCACCCTTACA

Mut= 86 bp

Wt= 101 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGTAGATGAGTTCATAGCTGCCATTAGGCCAGCTTCTCTTTCCCACCTCTCAGCTGGAGAGACCCACCGTGGTACCTGGAAACCTTGTGGGAGTAGCCTCTCTCCCTGAAACCACAGAGAGTCCCACTTCTCAGTCAGAGCAAAGAGTTGCACATGTCTCTGCTGTTgctattcatggaagcatccaggatgtttctatgtgtcatgctgtattgttcactcctttggctatgtgacttaagttgtgcaacaggaaccaggcaaggtgaccatatgggtgtgctgctgtctagaaattgtacagtgacatggccagcgatgctcttgccagcttcctgaatgcttgtcactgcctcttacggaagtcacctgtcctcattagataagaaagcagagatcgacatcctggctgcagagtatatttccacagtgaagactctctcgtcagcccagcgtgtggagcacctccagaagatccagagcgcctacagcaagtgcaaggagtacagtgatgacaaggtgcagctggccatgcagacctacgagatggtgagtgcaggggccagtgcagcctcctgctgccttccgtgcccctgaagGAGAGTGTGCAAGCAAGTGTGTAAGGGTGCATGGAAATGTGTAAGGTGTGTAGAAATGTGTAAGGGTGTGTGAGAGTGTGTAAGGCTGTGTGAGAGTGTGTAAAGGTGCATGGAAATGTGTAAGGTGTGTAGAAATGTGTATCTGGAATAGGAGTTTG

This mutation is a 432 bp deletion beginning at Chromosome 1 position 93,811,567 bp and ending after 93,811,998 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43741 CCT ACG AGA TGG TGA GTG CAG Wild type Forward A
43742 ACA TTT CCA TGC ACC CTT ACA Common A
43743 TCC CAC TTC TCA GTC AGA GC Mutant Forward A
43744 Fluorophore-1 CCT GCT GCC TTC CGT G Quencher-1 WT Probe
43745 Fluorophore-2 ACA TGT CTC TGC TGT TGA GAG TGT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43741 0.40 uM
43742 0.40 uM
43743 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.