Protocol 33357: Probe Assay - Psmd6<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

>chr14:14119788-14119885 98bp CTGCAGATGAGAGGTGCTC CAGAAAGATTCTCTTATCCAGTGAG

Mut= 113 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case and insertions with carrots ^g^ (G insertion):

 TTGTCGCGGCTGGACTCCCGGCAAAGGCGCCGCGGCTTTAGTGATGCCTGTCGGTGTTTAGCTGCCGGCCAAGGGACAGTCAGGGTTCTGTCCCTAGTAGTCGAGTCGAATCGGTGTCCTTTCCCTGACATGAGGACTCCTTTGGAACAGGAGCCGACCAGAGCAAACTGAGGATTCGATTCCCT^tccccttggccttagtcgcgatcagacctgctaagatcggctctcaagcaggctcccagcttcaaaccttagccgtgctacccaggcccatagttgggaggcgtctgctagctgaccttcggtggggtgggaaaagcgagcagaagctaacaggatacctcataatgcgtgatttgtggcttttagatatggctccttattatgaagccttgtgtaaatccctcgactggcagatggacgtggatctcctgagtaaaatgaagaaagcaaacgaagaagagttgaaacgtttggatgaggagctggaggatgcagagaagaatctgggagaaagtgaaattcgagatgcaatgatggcaaaagcagagtacctctgtcagataggtgacaaggtcagtgagatgccctgcccgctctgggtaggtgacagggtcagtgagatgccctgcccgctctgggtaggtgacagggtcagtgagatgccctgcccgctctgcagatgagaggtgctccctattttctggaggtacagaccaatgttgtacatc^AGTGCAAATGAGTAATATCTCACTGGATAAGAGAATCTTTCTGTTTTAACTGGAAGGATAATCATATTTACCTGTACTGAAGAGTCAGTACAGGAACAGTAGATACATGACAATGTTGCAGCACCCAATTCCCTGCTCTTAACAGAGCAAGGTGTGCGTTAGTCTTCCTT

This mutation is a 548 bp deletion beginning at Chromosome 14 position 14,119,831 bp and ending after 14,120,378 bp (GRCm38/mm10). In addition, there is a single bp (G) insertion at the deletion site that will not alter the results of the exon deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41220 CTG CAG ATG AGA GGT GCT C Wild type Forward A
41221 CAG AAA GAT TCT CTT ATC CAG TGA G Common A
41222 TCC TTT CCC TGA CAT GAG GAC Mutant Forward A
41223 Fluorophore-1 CTG GAG GTA CAG ACC AAT GTT GTA Quencher-1 WT Probe
41224 Fluorophore-2 TCG ATT CCC TGA GTG CAA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41220 0.40 uM
41221 0.40 uM
41222 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.