Protocol 37842: Probe Assay - Pigz<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

>chr16:31944211+31944328 118bp AAGCTTGGAACTGGCTCACC AGTGGGAAGACCACAGTGC

Mut = 110 bp

Wt = 118 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CCCAGAACGCGAGCCTGCGCAAGCCTAAGCTTGGAACTGGCTCACCCCTGCCTCTCTCTCTGCAGAGGACATCCTGGgtgtgcaggctacacggccctgggaattttaccccagcagctcctgccgcactgtggtcttcccactgctgacctctggctctaccttctggttactcaggctctgggaagagctggggctctggcctggtctggtgagtggctacatgctgctggtggggccccgattccttctcaccgccctctcctttgccctggactgggctgtgtacgatctggccccgttgtggggggcagatcgttggaatgccttgtgcctgctgtctggttcctatgtcaccctggttttctacacaagaaccttctccaacaccatcgagggactgctcttcacctggctgctggtgctggtgtcccctggtgtggtaaggagtcctacatcaaagaagcctaccccaggtccacggtggcacagatatcttcttggtgttatcttggctgctggcttcttcaaccggccaacctttctagcctttgctctggcccccctctctctctggggcatccatagagcttcagaacttggtggcatcagggccctgatccaggaggccctggtgctgttgccaggggctgctcttgctgcagtgctgtgtgtagccacggacagctggtacttctccagcctctcgagatccacaggcgtctttctcacacctgccaacttcttgtactacaacctggacccccaaaaccttgccaggcacggcacacacgcacggctcactcacttggcagtcaatggcttcctgctctttggggtgttgcatgctcaagctctgcaggctgcatggcaacaactccacgcctgcctccgtgcatccccacaaacgggtctctccagggtgcggggtgcccggggtctgttgtctagctccaagtcttatctcctcctcttctactttacacccctgctcctgctttctgccttcagccatcaagaggctagatttctgattcccctcctagtccctctagtcctgctgtgcagtccacaaacccaacctataccctggaaaggcaccctggtcctcttcaacatcctgggcgctctcatctttggttgcctgcatcagggaggtctggtaccgggcctgaagtacctggagcagatagtccacacgcctgacctctcaggcacgaccacccactacacacttctcttcacacacacctacatgcctccccagcacctcttacacctctcaggcctgggatcacccgtggaggtggtagacatgggtggggctgaggacagggtcctgtgtcaagccctgaacaacttcagcagacaacctgcctgccaactggctggtgagccatggccctgtcggctctttgtggtaactcctgggaccaacaggcacgctctggagaagtgccgttttcccctcaagaatgagactctcttgtttccccacttgaccctggaggaccctccagccctgtcctctctgctcagtggggcttggaggaagcaccttagtctccatgtcatagaactggagacgcctgttgtgACAAAGCAGCCCAAGGCTCAGCCATAGAAGCCATTCCACCTTCTACACAGGCTGCTGGGCTGGGACATTGGA

This mutation is a 1520 bp deletion beginning at Chromosome 16 position 31,944,262 bp and ending after 31,945,781 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52306 AAG CTT GGA ACT GGC TCA CC Common A
52307 AGT GGG AAG ACC ACA GTG C Wild type Reverse A
52308 CCC AGC AGC CTG TGT AGA AG Mutant Reverse A
52309 Fluorophore-1 CCT GGG AAT TTT ACC CCA GC Quencher-1 WT Probe
52310 Fluorophore-2 CCT GGA CAA AGC AGC CCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52306 0.40 uM
52307 0.40 uM
52308 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.