Protocol 36103: Probe Assay - Zmat4<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

>chr8:23902255+23902354 100bp AGCTACAAGCTTTTTGGGTTAG CATGAACTCATACTCTAAAGGTCCT

Mut= 118 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (AGCAAG insertion):

 GTGAGGCTAAAATTGGCAAAGGCAGAGCTGAATGCCAAATGATAATTTCTGCTAAATAATTCAGGCCATGTGAACTTTGATATTTCCCTTACTTTAATGAGTATAATGTGGGCAATTACCAACTACTCTTCTCACAGAGCCAGACAATGTGCTTTTTCCTAGTTATGGGCTTCATTTTTCTTCGGGTAACTCATATCTGCAAAGTTTGCTATAAATGCAGGGCCTTGCTCATGCGACC^gctgcataaatgtatattactgaggtgatggtaattataacgaggaagtttagcatgtctgtaagctgttcatcaatagacaaatcttatgacatgggacttgagttcttctgtgacactgtctttctccttttccccatgcagggaagcgatgctgacatggtggataagaataaatgctgtacactctgtaacatgtcctttacctcagctgtggtggccgactcacattaccaaggcaaaatccacgccaaaaggttaaaactgttgctaggagagaaacctccactgaaaaccacaggtatgtatggaaagaaaaaaaatgatccaggctggtacagagaacctctcttttcagccataggaatcaacctctttttcagaagctacaagctttttgggttagatggcaggtgccattcaaaacc^CACAGGGAAGGATGGGAAGGCTGAGCCGAACAGGACCTTTAGAGTATGAGTTCATGGGCTTGTGCACTGTCTTTCATCTAATGCTTACAGCCTCCAGGATGTCTGTCACGTTGATTGCATATCATGTGAGATACACATCTCTAGTGCTCTTTCCAAGTTCAGCCCACAGTCCACATTGGTATGGTTGTGCACACTGCATAGGTAGTAGGTTCTTGCCAGTGTCTATGGAAAGCAATTAGCATCCTTGACATTTGTGCTTTGA

This mutation is a 428 bp deletion beginning at Chromosome 8 position 23,901,871 bp and ending after 23,902,298 bp (GRCm38/mm10). In addition, there is a 6 bp insertion (AGCAAG) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47844 AGC TAC AAG CTT TTT GGG TTA G Wild type Forward A
47845 CAT GAA CTC ATA CTC TAA AGG TCC T Common A
47846 CGG GTA ACT CAT ATC TGC AAA GT Mutant Forward A
47847 Fluorophore-1 ATG GCA GGT GCC ATT CA Quencher-1 WT Probe
47848 Fluorophore-2 ATG CGA CCC TTG CTC ACA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47844 0.40 uM
47845 0.40 uM
47846 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.