Protocol 36920: Probe Assay - Rfxap<em1(IMPC)J>Alternate 1
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

>chr3:54807127-54807276 150bp TACAAGAAGAAGAAGAGCGACCAG AGACCCTGAGTGTGTACCAAGT

Mut = 161 bp

Wt = 150 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GCTGGCCTCGAACTCAGAAATTTGCCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCATGCGCCACCACGACCGGCTGTCAGCCGGGTTCTTGGTCAGCCTGCACCTAGGAATCGCTTGCGGATGGCCGCCGCGCAGGCGCAGTCGGTTCTGTCTCGCACCTGCTCAACCCCGCCCCTCTCGCCGTTCCCAAGCAGAGGCTCGCGGACCGCTACGGCGCACGCGCAGTGCAGTGCTGGCGGTTCGGCTCCGGGACGTGCGGCGGGCTGCCGCTTGTCGAGTACGGTTGTGTTTCTCAAGAGCTGACGGGCCGCGGTGGCGGACCCGGGCTCGGGCGGCAGCACGGAGGCGCAGGCTGTGCccgagggctcggggccgagcaccgccagtccccggacggcccctccggtgacggtgctggtgatgcggcaggacgaggccgaggcggacggcgctctgaggccgggcctggcggggagtgaggcggcggcagacgccgaggacgaggcgggggacgacgacgccgacctgctggacacatcggacccggcgggcggcggcgagagtgcggccagcccggaggagctggaggacgaggacgcggagggtggcggcgcagcgcgcaggcgaggctccaagacctgcacgtacgagggctgccgcgagaccaccagccaggtggccaagcagcgcaagccctggatgtgcaagaagcaccgcaacaagatgtacaaggacaagtacaagaagaagaagagcgaccaggcactgggctctggcggcccgtccgcggccagcacaggcaacgtcaaacTGGAGGTATGCTTGCTCCTCGGGGCCCCGGGGCCAGGGCAGATGGGTGCCAACCTACTTGGTACACACTCAGGGTCTCCCCAAGGGCTCATCCTCTGCTCCAGCTGACATGGGCACTTTGGAGGTGATCTACACCTCTCCTTAGTAAACCCTTCCCAGTGTCTACCCCGAGGCATCCGGCACATTGACACACCCTGTTGGTTTTCTGTAACTTTGGTGACTTTGTTTTTTTTTCTTAGACTCCCTGGCCCCTGAGAAA

This mutation is a 453 bp deletion beginning at Chromosome 3 position 54,807,204 bp and ending after 54,807,656 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49103 AGA CCC TGA GTG TGT ACC AAG T Common A
49104 TAC AAG AAG AAG AAG AGC GAC CAG Wild type Forward A
49105 CGA GTA CGG TTG TGT TTC TCA Mutant Forward A
49106 Fluorophore-1 CTC TGG CGG CCC GTC Quencher-1 WT Probe
50046 Fluorophore-2 CGC AGG CTG TGC TGG AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49103 0.40 uM
49104 0.40 uM
49105 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.