Protocol 40093: Probe Assay - Gcn1<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

>chr5:115575875+115575964 90bp GTGTTCTTAACCCCTCTTAGCC AGGCCTCACTGGCTAAATCC

Mut= 90 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

The mutant probe (primer 55233) anneals over the nucleotide sequence containing mouse genomic variations rs49091205.

Sequence

Wt Sequence (deletion in lower case):

GTAGATCTGCCGGCCACTGCTGCGATTAAAGGTGTGCCTCACTACATGTGGCCTTCCTGCTGACTTGTAGACCACTTATTAGACTGTATGTGAGCAAGTACCTTCCGAGCATCACCAACAGCACAGTTTAAAGCCAGCGCCCAGGCCGGAGAGCAGAGCAGGCAGTCGTAGCCCTgctgagccctgctctgtagggagagggagatgctggggtcttgggcttcttctatccatcgttggtttagcaagtgtctcctgcactcggggcgtttctgttccatgtgcctgttttctccttgagattttacaaggtgcggggcttactagggcgtctggtaccaggatcccagctgacaggactgatgtaagcggacactcaccgctgtgcttgttgttgagactctgtgaactctgctttcagatctgccagagggagccgtgaaagggctctgcaagttgttctgcttgacactgcatcgatataggtgagcccccagagaccagggcttagaccgtgccatacagcgacactgtgtcagagagacagcatggggcagtggggagagcccctagttgttagagccagactcttaggttcatgctcttaactactggatcctgggcaagttacttagcctccctatgcctcagttttctcatctgcaaaatggggatgatagcattttgtacctcatgggaagttgttagtgtgaaggcctgggacactgcctgccttattataatatctcagcgcatgtatgcaatcattagctgcacttataaagctataaaggagtcatggggcatatacaccatgagcatagattctgcccttcttgtcatcctgtgctctttctggggtgaggtgggtttatcaggttcatctctgtcccctaggggcccctgaaatgtaggaccatgagtgtccccccgaagagcagtgcttgacttcagcagccctgtctctgctgaggtcacatgtcactaccccttccttccctctttagagacgcagcctctcgcagagccctgcaggcagccatccagcagctggccgaggcccagccagaggccaccgccaagaacctcctgcactctctccagtcttcaggggttggctccaaagcatgtgttcccaggtacgggcctggggctgcgggcctccggggggtggtgccaacttctcaggtaggctggtgtagagccactggtgggagcacatctggccctcttacctttgtactctgaatggcactagagagcgcctctgagtggggagaggaaccaagttagtagagaaaagatccagtgggcccgaggcactggatgggcaaagctttatatacttgttcatgtacattttatgtgaatgagtctcttactgcacgtatgtctgtatactatgtgcatgcctagaacctgaggataccaggggagggggtcaggtccccgggacagtgggagctgccatgtggttgctgtgagaatcgaacccaggtcctttggaagagcaacctgtgttcttgttttttggttttttgtttgtttgttttgttttttgtttttgtttttttcgagacagggtttctctgtatagccctggctgtcctggagctcactttgtagaccaggctggcctcgaactcagaaatccgcctgcctctgcctcccaagtgctgggattaaaggcgtgcaccaccatgcccggctgcaacctgtgttcttaacccctcttagccgtctctccaacctcatcttgtttttactgtcccaaacatcaagggatGGGATTTAGCCAGTGAGGCCTGGACGCTTGTCCTGTTTATCACTGATGGTGTTGGGGGAGGGTTCATACACACACACCATGCTGTGCCACAGGGTGAACATTGTTGTCCCCCCCCAATCCCCCGTGGAGGCTGGAACCCCGCACTAAGCTCTTCTCCTCCCGCTTCTCCTTTGTAGCAAGA

 

This mutation is a 1611 bp deletion beginning at Chromosome 5 position 115574333 bp and ending after 115575943 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
55229 GTG TTC TTA ACC CCT CTT AGC C Wild type Forward A
55230 AGG CCT CAC TGG CTA AAT CC Common A
55231 GAG CAT CAC CAA CAG CAC AG Mutant Forward A
55232 Fluorophore-1 TGT CCC AAA CAT CAA GGG AT Quencher-1 WT Probe
55233 Fluorophore-2 CCG GAG AGC AGA GCA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
55229 0.40 uM
55230 0.40 uM
55231 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.