Protocol 33361: Probe Assay - Rnf123<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

>chr9:108070997-108071082 86bp TCATCCTGATGGAGGTAGCC TCAGGAATATGTCAGGGGTGT

Mut= 85 bp

Wt= 86 bp

Fam=Mut

Hex=Wt

Sequence

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

GATGATTGTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGGACCTCTGGAAGAGCAGTCAGTGCTATTAACGACCAAACCATCTCTCCAGCCCCAGAAATCACTTTTTCACTGAGGAAGGTTGAGACCTG^ctggtctggagcttcagccaagcatcctccagtgttcaaggagaagggtgacagggacttactgtttggctgagctcaagagcttgtgggtccccagcatctctcattccctaggaaaccgctaaacttccataacctgcctgagcacgtggaccagctgctacaggtggacagtgaagacaacgagagccagggtaggaggtgttctcaggggtggggtgggtgaggaatgtggtgtcaaagcctggagttctctgcacagctcatgtctccctgtgtctgtcagaccctctgccatccctgatttcacacatctcccttcccaggacaagttgaaggtcgacttggcccatctactgtggtcctagaccacacaggaggctttgaggggcttctccttgtggatgatgacctcctgggggtgagtggagatgtgtcttgggccccagctcagccccatcctctacgcttatcctgatggctacatgttcatcctgatggaggtagccatacacctatcttgacaccactccaag^CCATGGTGTCAGCCCTTAACACCCCTGACATATTCCTGACACCATGATACAGTCACTACCTCCAACCCTTTCAGCAAAGCCCAGAGTCCCATCATGTCTGTCCCAGAAGCTCTGGACATGGATACAGTTTCTCCTGCCTCAACCAGACACAGTGCAGTGTTGGCCATGACAG

This mutation is a 536 bp deletion beginning at Chromosome 9 position 108,071,036 bp and ending after 108,071,571 bp (GRCm38/mm10). In addition, there is a 47 bp inverted sequence from the deleted region Chr9:108071521-108071567 (TCACCC through TCCAGA) at the deletion site that is not predicted to alter the results of the exon deletion.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41234 TCA TCC TGA TGG AGG TAG CC Wild type Forward A
41235 TCA GGA ATA TGT CAG GGG TGT Common A
41236 CAC CCT TCT CCT TGA ACA CTG Mutant Forward A
41237 Fluorophore-1 ACA CCT ATC TTG ACA CCA CTC CA Quencher-1 WT Probe
41238 Fluorophore-2 AAG CTC CAG ACC ATG GTG TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41234 0.40 uM
41235 0.40 uM
41236 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.