Protocol 45340: Probe Assay - Guk1<em1(IMPC)J>Alternate1
Version 2.0

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

>chr11:59186041-59186161 121bp TGGGGACAGTGGTATCTGTTC GGAGAAGAAGAGAGCTTTCCAG

Mut= 106 bp

Wt= 121 bp

Fam=Mut

Hex=Wt

The WT Reverse primer (primer 65355) anneals over the nucleotide sequence containing mouse genomic variations rs214496484.

Sequence

Wt Sequence (deletions in lower case):

CCAGCAGGAGAATCTCTCTTGCTGTTGGGTTCACAGCTTGATTAGAGTCTGCATCATAGTGAAGGCTGAACCTTCTTCCTGCCTGGTGTATGGAACCTGGCCATTTCCAATCCTTCAGCAAAGACAATGGGGACAGTGGTATCTGTTCCAGCCTAGATCTGGCCATAGCAGTCCTGTgtagggtggagaggggccaggccaggcagggatgggttttgggtaccttctggaaagctctcttcttctccagattactactttgtgaccagggagatgatgcagcgtgatattgcagcaggggacttcattgagcatgctgagttctcagggaacctgtacgggacaaggtgggctgagtttggtgtgggcccagcttcatggatcagggtaattccagaactgttaacaaaagaaagcactctagcatgtcacctctgacctcacagtgccctcccagtctcccgcctagggctgggatggcttgctaaccccctagtctctgagcatagctgataggctccagggaacaagcaggcccagtcatgccccaggctcatctgtcctcaagccatctggcgaggcaaagagccacatgaggcacctcacGTGGAGAGGTAGGCAGTGCCAGGCAGCCCAGGCCCAAACCTTTTTGGCTTTCGGCTACAGGACAGGTACAAAGGGCTGTGATGTCAGAGGGCCTGACAGGGTTGACTATATGCTTGGAAGTTCTGGAAAGTACAGAAGGTGCTGGGTTCACGAGAGCCTCTGCACTACCCAGGCCAGGCCCAGAATGGGCCTTGGGGTGGAAAGGGC

 

This mutation is a a 429 bp deletion beginning at Chromosome 11 position 59,185,683 bp and ending after 59,186,111 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64355 TGG GGA CAG TGG TAT CTG TTC Common A
64358 Fluorophore-1 AGC AGT CCT GTG TGG AGA GGT AGG Quencher-1 MUT Probe
65355 GGA GAA GAA GAG AGC TTT CCA G Wild type Reverse A
65356 AGC CGA AAG CCA AAA AGG T Mutant Reverse A
65357 Fluorophore-2 CAG GGA TGG GTT TTG GGT A Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64355 0.40 uM
65355 0.40 uM
65356 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.