Protocol 37358: Probe Assay - Gm5127<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

>chrX:106709641+106709784 144bp TTTTATGAGGCCACAGTGCT GGGGCCATTATATCTCACAGG

Mut = 143 bp

Wt = 144 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

ACAAAACTATATTTCAAGTGCTTCTCGTTGTTTTGCATGCAATCTTATATCATTTTTTTCTTCTCTACTTTACTCCAGGAATCCTGCCCAACTTCACATTTTAAAAAATCGAGACCATAtgaaagacctccgcaggtacctgatgttcaaatacatccagtatctagtcctgccttctccgaatgtcatcatcattctgctgggatctcttgccagtctctatgtcatgctgctattgacatttccttccgtttccagaaaatccactgcggtgtttattggtagcatagctcaggcagacatcttagttgtctgcagcttgttttctgctatctcagcatgcgtgataaggagcgagccatcgtcaacttcttttcaactagccttgaggcaaaacttccaaattgcaaatatccatgccagttcccttttactcagctgtgttacccttgaggcttttctgattacttttcttccagtagagacacgccacataaggaatgttagatgtgccagagtggcttctaaaatcatctgggctgttgtaattaccgagtgtttcctttatcagcttgaatatgtcaaaggccttaacatctcctatcttggcattcataggcaaattcagctattgatgaatttcttttatgaggccacagtgctgctgaaattacttatttatcccattggagttcttctaagaatcttcaacgtatatcttttttataaaatgtatttccgagaccatTATAGTTAGTTTCTCAGGCCTGTGAGATATAATGGCCCCGTTGTTTTGTCAAACAGTCATATTTATGGGGCTATGAAGACATTTTATAGCTGTGGTT

This mutation is a 629 bp deletion beginning at Chromosome X position 106,709,117 bp and ending after 106,709,745 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51149 GGG GCC ATT ATA TCT CAC AGG Common A
51150 TTT TAT GAG GCC ACA GTG CT Wild type Forward A
51151 AAG TGC TTC TCG TTG TTT TGC Mutant Forward A
51152 Fluorophore-1 CCC ATT GGA GTT CTT CTA AGA ATC TTC Quencher-1 WT Probe
51153 Fluorophore-2 CTT TAC TCC AGG AAT CCT GCC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51149 0.40 uM
51150 0.40 uM
51151 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.