Protocol 48446: Probe Assay - Pbx4<em1(IMPC)J>
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

Mutant=  106 bp

Wild Type = 122 bp

>chr8:69864218+69864339 122bp TTTAAGTGATGCCCAGCCAT TCCTCTTCCTGAAACTGGAT

Sequence

Wt Sequence (deletion in lower case):

TGTCCCAGTGGGTCACTAGGTGTCTCTGATCCAATGTGGAAATAAAGAAACTGGAGGATGGCTTAGAGGTCAAGAGTGTTTCCTGGTCTTGCAAAGGACTGAGTTCAGTCCCCAGCACCTACACTGGGCAGCATACAGTTGCCTGTGAACCCAGCTCTAGGGGTCAGCCATGGACTTCCTGGACTCCCAGGGCACCTGCAGTCACACATGCACATGCCCACACACAGATGCGCACATAATTGATATTTTTAAGTGATGCCCAGCCATGGTGCCAGTGCCACTTGgtcctggctacttttctcttcagagagcacatcctggtctgcctctgttccctctagccgtgagcatccagtttcaggaagaggaccctcccgatgcccagctgctgaggttggataacatgctgctggctgagggtgtgtccaggccagagaagagaggccgaggagcagcagcaggcagcacagcaacaccaggtggctgtccaaatgacaatagcattgagcactcagactacagggccaagctgtcccagatccgacagatttaccactctgagctagagaagtatgaacaggtgatctttctacatgcaagagtttgtcttgtgaatgtgcactggccacagacagatgtggacatgagctccctgctctatggcagagcagggacagcccgccacagtcagtcactgcagaggggctggtgtcactgaaagcgctttcccctcactcctcagagctgacctgagtgtgagctgtgggggtggcagatgtggtcagctgtggagcccactgccccgcaccactggctgctgagtcacaggctggcccatcttttcccaccccaggcctgtcgtgagttcaccacacatgtcaccaaccttctccgggaacagagtagggtgaggcctgtgtcttgcagggagatggaacacatggtgaacaccatccagagcaagttcagtgccatccagaggcagctgaagcagagcacgtgtgaggctgtgatgactctacgctcacggttcctggatgccaggtcaggggctgcgtgccactggggtcccCGAGGGTTGGCCCTTAGATAATAGAAGCAGGATCAGAGTAGGATAGTTCCGATGTTTATGTCATAGTGTTGCAAGCGCTAATCCCATGTTCTCCCTACCACTGAGCCCAGCCAAGCTCTGTCTTGTTTTTAAGTCTGTCCAGAGGGTCTAGCCAAGGCCTCACATAGCTTCTTTATGAGAAGACCCAAAGTACAAACTGCTGGTTTGT

 

This mutation is a a 788 bp deletion of region Chr8:69,864,255-69,865,042 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
71825 TTT AAG TGA TGC CCA GCC AT Common A
71826 TCC TCT TCC TGA AAC TGG AT Wild type Reverse A
71827 ACA CTA TGA CAT AAA CAT CGG A Mutant Reverse A
71828 Fluorophore-1 TCT TCA GAG AGC ACA TCC TG Quencher-1 WT Probe
71829 Fluorophore-2 TGC CAC TTG CGA GGG TTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
71825 0.40 uM
71826 0.40 uM
71827 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.