Protocol 42141: Probe Assay - Rnf126<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

Mutant=  138 bp

Wild Type = 139 bp

chr10:79761705-79761843 139bp GCCTCTGCCTACCAGGAATC CCTCCAATGCAGTAACCAGA

The common primer (primer 59950) anneals over the nucleotide sequence containing mouse genomic variations rs240476437 and rs223341358 and the wt reverse primer (primer 59952) anneals over the nucleotide sequence containing mouse genomic variation rs222710171.

Sequence

Wt Sequence (deletions in lower case): AGCACTCCCAATTCCGAATGGAAGTTGGATAGTTTAGGCTCCACACCCTTGGGTCATGCATGCTCCATGGCCTCTGCCTACCAGGAATCCCTGTGAAATAGTTGTACCCAATGgcttcctcaccattggtagaagagcacgtgggtggtggaaacagaacaatcactgccagtggaaggtcagcttggtctggttactgcattggaggccagtgtgggctgcagagcaagcctatcttgtgaggtggtctgcaggcccgtgacactgtctcttccacagGATCATCCAGCAGCTCGTGAATGGCATCATCTCTCCGGCTGCTGTGCCCAGCCTGGGCCTTGGTCCCTGgtgagtggggggcgggtgcccggcccctcctaccctgcgccccctgacccggcctctccccccagGGGCGTCCTGCACTCGAACCCAATGGACTACGCCTGGGGGGCCAACGGCCTGGACACCATCATCACGCAGgtacagcggccgctcaacatctgtggggggccgagaggctggcctagtaaagcagatggggtgctgctctgtagcaccagaggcctctgcagctatctcgttctctggtgtcacacagctctggccaagcccttcttcccctcacctcctgaagaatgcctgctgtcctagggtgcacagtggcctgtaccagagctgcacacccctgatacccctgAGGTTTAATGGGCCTGGTCTTGCTGATTTGCAACTCTGCAGAGCTGGGCAGGTGGAGACTCCCCACCCTACCACCCCATTGGGAACCATGCCCTCCCCAC

 

This is a 587 bp deletion beginning at Chromosome 10 position 79,597,047 bp and ending after 79,597,633 bp (GRCm39/mm39). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
59950 GCC TCT GCC TAC CAG GAA TC Common A
59951 Fluorophore-1 ACC ATT GGT AGA AGA GCA CGT G Quencher-1 WT Probe
59952 CCT CCA ATG CAG TAA CCA GA Wild type Reverse A
59953 Fluorophore-2 TGT ACC CAA TGA GGT TTA ATG GG Quencher-2 MUT Probe
59954 AGG GCA TGG TTC CCA ATG Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
59950 0.40 uM
59952 0.40 uM
59954 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.