Protocol 41359: Probe Assay - Rassf10<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

Mut = 89 bp

Wt = 98 bp

Fam = Mut

Hex = Wt

>chr7:112554848+112554945 98bp AGGACTCAGATACTGGGCTGAG GCTTGCTCCCTACTACTTCTACA

Sequence

WT Sequence (deletions in lower case):

TCGGGAGCTGCCAGGTTGCCCGCGCTGGCCCTGCTGCCCTAATTGCACCTAGCCGGACTTGCTCCTCACGTCCCGGTTGCGCCATGgatccttcggagaagaagatatcggtgtggatctgccaggaggagaagctggtgtccggcctctcccgccgcaccacctgctcggacgtggtgcgggtgcttttggaggacggctgccggcggcgctgcaggcagcggcggggccagcgacggggcttgacggaagacccttcgggccagttggagctgcccgagcccccggacgaaaacgacgaggatgacgacgacgcgatgcccccgggcatgctatgtgggcccccgcagtgctattgcattgtggagaagtggcgcggctttgagcgcatcctgcccaacaagacgcggatcttgcgcctctggactgcttggggcgacgagcaggagaatgtacgcttcgtgctggtgcgcagcgaagcgtcgctgcccaacgccggaccgcgcagcgccgaggcgcgggtagtgctcagccgcgagcgcccctgcttggcccggggcgccccagcacggcccagcctggccttgacccaggagaagcagcggcgggtggtacgcaaggccttccgcaaactggccaagctcaaccggaggcgccagcagcagccgtcgtcaccttgttcgtccacgtcgtcgtccaccgcctcgtcctgctcgtcgtctgctcggacccacgagagcgcgtcggtagaacgtatggagacgctggtgcatctggtgctgtcgcaggaccacacgattcgccagcaggtgcagcggctccgggagctggaccgcgagatagaccgctacgaagccaaggtgcacctagaccgcatgcggaggcacggagtcaactacgtgcaggatacctatctagtgggggccggcatcgacctggacggacaaactcctgaaggggagccggaagatgcgacactggaggagaaggggacagaaccggcggcacccctggacagcgaggcgcaggcggcagcgttggaggagctggccaggcgctgtgacgacctggtgcggctgcaggaggagcgcgcccagcaggaggagttgctagagcgcctgtccgccgagatccaggaggaactgaaccagcgctggatgcagcggcgcaatgaggagctggcggctcgagaggagtccctagagcccgatggtggtccggatggcgagctgctgctggagcaggagcgggtcaggacgcagctcagcaccagcctttacatcgggctgaggctcagcacggacctggaggccgtcaaggcggacttggattacagccagcagcagagggacattaaggagcgcgagcttcagggccttctccagagtttgcacacttttgagcagacggtggtgcatgatggggctctgggttccagcggaccctctcgcgaacctcagcctcagacctgtgcagaaatgtgggtagaccaggcccggggactggctaagagttgcccgggcaacgatgaggactcagatactgggctgagctccatgcatagccaagactcagactcggtaccccctgtgtgtgAATCCCTTGTGTAGAAGTAGTAGGGAGCAAGCTTAGACGCTTGCAGAATGCACTGGTTTGGCCTGCTCAGG

This mutation is a 1510 bp deletion beginning at Chromosome 7 position 112,553,404 bp and ending after 112,554,913 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58137 GCT TGC TCC CTA CTA CTT CTA CA Common A
58138 AGG ACT CAG ATA CTG GGC TGA G Wild type Forward A
58139 CCT GCT GCC CTA ATT GCA C Mutant Forward A
58140 Fluorophore-1 AAG ACT CAG ACT CGG TAC CCC CT Quencher-1 WT Probe
58141 Fluorophore-2 CCG GTT GCG CCA TGA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58137 0.40 uM
58138 0.40 uM
58139 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.