Protocol 43270: Probe Assay - Gm14569<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=  106 bp

Wild Type = 125 bp

Fam=Mut

Hex=Wt

X-linked

>chrX:36479941-36480065 125bp CCTTCTGAGCTCTCTATGGTATCT GACTTGACTCCCATGCCACT

Sequence

Wt Sequence (deletions in lower case):

TGTGTATGTATGAGTGTGTGTGTAAGAGAGATGTGTATACGTTGACTGGGTCCACACTCTTTTTTTTATTCTCTCTCTCCATCTCAGAGATGGCTTTCAGTGTCAGCCCATCTGCATCTCTGCATCCTTCTGAGCTCTCTATGGTATCTATGTTATGAAATACCTCACCCTTCAattcttaatccctcgcactgcttttacaagacttttcctttccacataggccaaagagtggcatgggagtcaagtccatatcccatgacagtgtcttctgtttggatcctgagcctgaaaaaggagcaggcaagctacactcttctccagctccccacagaagcaaatcactaaaggtaacaatgagtctgacctcactctgattagacactgttctatcctgatcccttctCAAAGGAGGGAGGAATTTGATCCTGATTATCAACTAATTCTCTCTCACAAGACTATCTTAAATTTCAAATAATACAATATATAAATAGGCATTTAACTATGTGACATGATGAACACATAGTGTTGGCTGTTGTGTGCTTTTATCTATCACACCCAAAGATACCATAATTATTTTGATAATGGACAGTTTACTCTTTTTTGTTGTTGTTGTTGGTTCTTTTTTGGAAAGTTTACT

 

This mutation is a 232 bp deletion beginning at Chromosome X position 36,479,785 bp and ending after 36,480,016 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
62635 CCT TCT GAG CTC TCT ATG GTA TCT Common A
62636 GAC TTG ACT CCC ATG CCA CT Wild type Reverse A
62637 GAT AGT CTT GTG AGA GAG AAT TAG TTG Mutant Reverse A
62638 Fluorophore-1 TTC CTT TCC ACA TAG GCC AAA G Quencher-1 WT Probe
62639 Fluorophore-2 TCA CCC TTC ACA AAG GAG GGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
62635 0.40 uM
62636 0.40 uM
62637 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.