Protocol 41677: Probe Assay - Gmeb2<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

>chr2:180902350-180902466 117bp ATACTTTTGTGGGGCGTAGC CATGCTCTATCAGCCACAGG

Mut= 157 bp

Wt= 117 bp

Fam=Mut

Hex=Wt

The wt reverse primer (primer 58900) anneals over the nucleotide sequence containing mouse genomic variation rs250950845. The mutant reverse primer (primer 58901) anneals over the nucleotide sequence containing mouse genomic variation rs253270638

Sequence

Wt Sequence (deletions in lower case):

TAGCAAACCATAGAACACTGTTGAGTCAAGAATGTGGTTCTCCTGACTTTGAGGTGGACATGTGTAAGCACTATGCAAGACAGAGCACTGTGCATTTTACATTTCTGTGGCTTCTTAGTGGGCTCGTCCAGACAAGGTATCATCCATTGGCAGGCTATATGCTTGTGTTTTGATTAATATCATGTTTTTCTGATATGATATGGGTGAATATCACTAAAGGAATAAGTTATCCATAAGAAAGAGATTTGCTGTGTTCTTTCTAGTATAAGAGTACATAAGCTCTCTTCTCTGCTGTGGTTTGACCCTCATCTCCTACATAATATACTTTTGTGGGGCGTAGCATGCCAAGAATCCGGTAGAGaactggctactggggatattccaatagagcactatatcagagtgttctatactgagacctgtggctgatagagcatggttgtctttcttgctgtgaacctctagtgcaggctggagcaggaagggagaacaggctgtgctgcagtgtcaccaggcctgtgtcaggctgagaaggtcctctactgtcaccaggcccgtgtcaggctgagaaggtcctctactgtgtttctttctagtatgatgaacatgtgatcagcccaaaggagtttgtgcacctggcagggaaatctaccttgaaggattggaagagagccatccggatgaatggcatcatgctcaggtgggcatctgacgagtctgagatGATGGGAGGGGTTTGTAGCCATTGCTTTTTCATTTGATTTTAGATTTGTGAAGATGGAGCATTTTAGTTGTTTATTTTAATTTCCACTAATTCTTTACTTGGTGGTAGTGGTGATGGTGATGTGTTTATAATTTGAATATGTCACTGTTAAAACTTAAGGATATAATCTGGTGTTTGTGCATAGATAAACATTTAGGCTTACTTTTTTAACAGCTCTTTATGCTACCCTTTACTTCTGTCTTTCATTAAAGTCCCTTGCAAAGTGCACA

 

This is a 363 bp deletion beginning at Chromosome 2 position 180,902,064 bp and ending after 180,902,426 bp (GRCm39/mm39)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58899 ATA CTT TTG TGG GGC GTA GC Common A
58900 CAT GCT CTA TCA GCC ACA GG Wild type Reverse A
58901 CCA TCA CCA CTA CCA CCA AGT Mutant Reverse A
58902 Fluorophore-1 ATC CGG TAG AGA ACT GGC TAC TGG Quencher-1 WT Probe
58903 Fluorophore-2 ATC CGG TAG AGG ATG GGA GGG GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58899 0.40 uM
58900 0.40 uM
58901 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.