Stock No: 000528
Protocol 32966: Probe Assay - Phex<Hyp> Probe Alternate1
Version 2.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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=  159 bp

Wild Type = 190 bp

>chrX:153596983-153597172 190bp TCCTCAAGCTTATTTAGTGCCTTAC TGTCCTTCCATGCCCTGA

Sequence

Wt Sequence: atgccaagagtcgaataaacatttcttgggtgcaaaggtgttgtgaatggaaaatattacaaagcccagatctagaacatattcctatttaatatttaaggaaattggaataatccttatttcttggtatccaaagctgtctgaaactcctttccatactaaggtaaagcttagatatatacccaatgggaATATATATATATATATATATATATATATATATATATATATATATccaatgggaatatatatcagaatatgagttctgagtctaggtaataaaagccattatgacttccaacttgcttttcttgatttatcactctgggagaagccaatcaccatgtcattgggccacttaagcagattgtggataggcccacacgagaggctccagagggcttctgcccatttctagtaccatgcagatcctcaagtccaagcatttgggtcagtacaacgaattccttcattttgactgcagtcttccaagagaccttcagacagaccattcccccaatagctgcttccaaattcctggaccagagaaaatgaaaaa

 

Mutant Sequence: ATGGAAAATATTACAAAGCCCAGATCTAGAACATATTCCTATTTAATATTTAAGGAAATTGGAATAATCCTTATTTCTTGGTATCCAAAGCTGTCTGAAACTCCTTTCCATACTAAGGTAAAGCTTAGATATATACCCAATGGGAatatatatatatatatatatatatatatatatatatatatatatGTATATGGTGTGTGTGTCCAGTATTATTAGCatAAGAGCCATAGCTACATTGAAGAAACTGAGGTTCAGAGCTATAAATTATTGTAGGT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35058 GCT GTC TGA AAC TCC TTT CCA Mutant Forward A
35059 GTT TCT TCA ATG TAG CTA TGG CTC T Mutant Reverse A
35060 TCC TCA AGC TTA TTT AGT GCC TTA C Mutant Forward A
35062 Fluorophore-1 TGG TGT GTG TGT CCA GTA TTA TTA GC Quencher-1 MUT Probe
35063 Fluorophore-2 AGG AAC TAA GGC TCA GCA GAC AG Quencher-2 MUT Probe
40367 TGT CCT TCC ATG CCC TGA Wild type Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35058 0.40 uM
35059 0.40 uM
35060 0.40 uM
40367 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.