Protocol 34147: Probe Assay - Gk2<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

>chr5:97456904-97457011 108bp GCATCACCAGGGAAACAGTC AAGCGAGTGGAGTTGGTTC

Mut= 100 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGACTTAGGTGAGGGGGTAAATAGGCTCCCGAAGCTGTGGGACTGGCAGCTGCGCCTGCGCACTCCACCGTACCTGACTGGCTGTTGTGTCTCCTAACTGGCATCACCAGGGAAACAGTCCACTGACCAGTTTCATGGCAGCCTCgaagcaaacctctgcgggaccgttggtaggagcggtggtccagggaaccaactccactcgctttctagttttcaatagcaaaacagcagaactagtttgctctcatcaggtggaactgacacaagaatacccaaaagaaggatgggtggagcaagaccccaaggaaatccttaaatctgtctatgagtgcatagcgaaagcttgtgaaaaacttgctgaagtgaacattgatatttccaacattaaagctatcggcgtgagtaaccagcgggaaaccactgtggtctgggacaagttcacgggagaccctctgtacaatgccgtggtgtggcttgatctcagaacccagtctactgtggagactctcactaagaaaatcccaggaaatagcaactttgtcaagtctaagaccggccttcctcttagcacttacttcagtgctgtgaagctgcggtggatgctggacaacctaagacccattcaaaaggctgttgaggaaggcagagccatgtttggaaccatcgactcttggctcatctggtgtatgaccggaggagtcaatggaggcatccattgcaccgatgtaacaaatgcatgcagaacgatgctttttaacatccactctttggaatgggataaagatctctgtgacttttttgaaattccaatgagcatcctcccaaatgtatgcagttcttctgagatctatggcctgatgacatcaggggccttggaaggcgtgccaatatctggatgtttgggggatcagtctgctgctttagtgggacaaatgtgtttccatgaaggtcaagccaaaaacacatacggaacaggctgttttttactatgtaatacaggccagaaatgtgtgttttctgaacatggccttctgaccacattggcttacaaactaggcaaaaataagccagttttttatgccttagaaggttctgttgctatagctggtgctgttattcgctggctaagagacaatttcgaaattataacaacctcaggggaagttgaaaatcttgcaagagaggtaggcacttcctatggctgctactttgtcccagccttttcggggctgtatgcaccttactgggaacccagtgcaagagggataatctgtggtctcactcagttcaccaacaaatgccatattgcctttgctgcgttagaagctgtttgtttccaaacccgagagattttggatgccatgaatcgcgactgtggaattccactcagtcatttgcaggtcgatggaggaatgaccaacaacaggattcttatgcagctacaggcagatattctgcacattccggtagttaagtctgtcatgcctgaaacgacggccctgggagctgccatggcagccggagctgcagaaggggtaaatgtttggagtcttgaacctgaagatttgtctacgatcttgatggaacgatatgaaccacaaatccaagccacagagagtgaaattcgtttttcaacatggaagagagcagtgatgaagtcaatgggttgggttacagcaaaggaccctgaaaatggcgataaccctgtcttctcttgtctgcccttgggtttttttatagtgagtagcatgacgttattaattgGAGCAAGATGTGTCTCAACTGCTGAAGAGTAATTTGAACCACGGACTTGCAATGTGTGAACTTTTTACAGAAAGAATTCCATCTTTTAGTGTGAAACCAATATTATAGGTTTATTTTTACCTATAGGCCACTTGCTTACAGGAACC

This mutation is a 1622 bp deletion beginning at Chromosome 5 position 97,455,345 bp and ending after 97,456,966 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42919 GCA TCA CCA GGG AAA CAG TC Common A
42920 AAG CGA GTG GAG TTG GTT C Wild type Reverse A
42921 ACA TTG CAA GTC CGT GGT TC Mutant Reverse A
42922 Fluorophore-1 ACC GTT GGT AGG AGC GGT Quencher-1 WT Probe
42923 Fluorophore-2 AGC CTC GAG CAA GAT GTG TC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42919 0.40 uM
42920 0.40 uM
42921 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.