Protocol 41454: Probe Assay - Zfp146<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

Mutant=  94 bp

Wild Type = 124 bp

chr7:30161703-30161826 124bp GGTAAAAAGCCTTATCAGTGCAG GAACACTTCCCCGTACTGAAG

Sequence

Wt Sequence (deletions in lower case)

AGCTGCAAAGCCCTGAGCCGGATGGCGGCGCGCTCGGCACTGCTGGAGGAAAGCAAGTGAACTTCGCAGAGACAACATTGGCTATCAGTTAAACTTGGCCTGGGAAGAACGTTTGTTCTGATAAAGCTCTCTTAAACCTCCTCCCTTGCTCTGCATTTGAGTGAGCAGGCAGGCACAGAAAGAAGCCCTCGGACTGTAAGAAATGTGGAAGCATCCTCAGTCAAGAGCAGAGCCTCGTTTGTCTGGACTTCCCTATTGCAGAGAAGCGCTGTGAATGAGATCACTCTTCCACTCAGATGTCACAGCTCAGCCAGCAGAGAATCttaagcgggggaagcccctttgcctgtaaggtatgtgggaaactcttcagccacaaatcgaatcttacagagcacgaacatttccatagtagagagaaaccgtttgagtgtaacgagtgcgggaaagcctttagccagaaacagtatgtcatcaagcaccagagcactcacagcggagagaagctattcgagtgcagtgactgcggaaaagcctttagccagaaggaaaacttgcttacgcatcagaaaattcacactggagagaaaccttttgagtgcaaagattgtgggaaagccttcattcagaagtcaaacctcatcagacatcagagaactcacacgggggagaagcccttcatatgtaaggagtgtgggaaaaccttcagtggcaaatccaatctgacggagcacgagaagattcacatcggagagaagcccttcaagtgcaacgagtgcggaacggcttttggccagaaaaagtacctgataaagcaccaaaacatccacaccggggagaagccgtacgagtgcaacgagtgcggcaaggccttctcgcagcgcacgtcgctgattgtgcatgtgagaatccattcaggcgataagccttacgagtgcaatgtatgtgggaaagccttctctcagagctcatctctaaccgtgcatgtgaggagccatacgggtgagaaaccctacgggtgcaatgaatgtgggaaggccttttctcagttctcaaccctggccctgcatctgagaatacacacaggtaaaaagccttatcagtgcagtgagtgtgggaaggctttcagccagaagtcccatcacattagacaccagaaaatccatactcattaaacgCCCTGAATATCTTCAGTACGGGGAAGTGTTCATTAGGAATTTATACCTCATAGCGCATCGTTTGGTCACTCTGGCTAAGCACCATAAATCAGCCAAACATTTAACAGAAGATGTCGTGAACACCGGAGAATTCACAATGAAGGGAAAGACATGTGTATGATACAGGCCTGCAATCAGCGAATGCAGCGGTGGAAAAGCAGAGCAGTGCTGAACTGTGAACACAGTCGTCTGCGGTCAAGAGTGGCAGGCAGGCAGGCCTGTTGTGA

 

This is an 855 bp deletion beginning at Chromosome 7 position 29,861,159 bp and ending after 29,862,013 bp (GRCm39/mm39). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58397 Fluorophore-1 AGC CAG CAG AGA ATC CCC TG Quencher-1 MUT Probe
58398 Fluorophore-2 AGT GTG GGA AGG CTT TCA GC Quencher-2 WT Probe
58399 GAA CAC TTC CCC GTA CTG AAG Common A
58400 GAG AAG CGC TGT GAA TGA GA Mutant Forward A
58401 GGT AAA AAG CCT TAT CAG TGC AG Wild type Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58399 0.40 uM
58400 0.40 uM
58401 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.