Protocol 31647: Probe Assay - Dtx3<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

>chr10:127192598-127192705 108bp GATGCTGGTCTCCAAGGATG TGATCTCTTACCCCCTGGAC

Mutant=  109 bp

Wild Type = 108 bp

Sequence

Wt Sequence: GAAACAGAGGTAACACAGGGGGTTCAGAAGGAGCCTGAAGTAAGAGGCACATTTATCTGTCAAATGGGGACTCCCCACTCTGAGTCCCTTTCACCCTGGGGGCAGTgtcgttcgtcctgtccagaatggcagcctgtggaggctcctgcaagaacaaagtgactgtctccaagcctgtgtgggacttcctgagcaaggagaccccagcccggctggcccggcttcgggaggagcatcgggtgtccatcctcatagatggcgagacttctgacatctacgtgctccagctgtccccacagggtcctcccccagcccctcccaatgggctctacctggccaggaaggctctcaaggggctactgaaagaggcagagaaagagctaaagaaagcccagaggcagggggagctcatgggctgcctggctttggggggtggaggggaacaccccgagctgcaccgcccaggccctcctcccctccgagcagccccactcctgcccccaggggcaagagggctaccccctcctcctcctcccctgccccctccactccctcctcgtctcagagaggacgctgaagagcaggaaaccacctgccccatttgcctgggggagatacagaatgccaagacgctggagaagtgcaggcactccttctgtgagggctgcatcacacgggccctgcaggtgaaaaaggcctgccccatgtgcggccgattctacgggcagcttgtgggcaaccagccccagaacggacggatgctggtctccaaggatgccaccctcctgctgcccagctacgagaagtatggcaccattgtcatccaatatgtcttcccgcctGGCGTCCAGGGGGTAAGAGATCACAGTTCCTACCCGTTGGCTTCCTCCAAGTTCACTGTCTCTTGACTTAAGAAAAAAAGGCAATGTAGAGAGTATGTTGTTAG

 

Deleted Sequence: GAAACAGAGGTAACACAGGGGGTTCAGAAGGAGCCTGAAGTAAGAGGCACATTTATCTGTCAAATGGGGACTCCCCACTCTGAGTCCCTTTCACCCTGGGGGCAGTgtcgttcgtcctgtccagaatggcagcctgtggaggctcctgcaagaacaaagtgactgtctccaagcctgtgtgggacttcctgagcaaggagaccccagcccggctggcccggcttcgggaggagcatcgggtgtccatcctcatagatggcgagacttctgacatctacgtgctccagctgtccccacagggtcctcccccagcccctcccaatgggctctacctggccaggaaggctctcaaggggctactgaaagaggcagagaaagagctaaagaaagcccagaggcagggggagctcatgggctgcctggctttggggggtggaggggaacaccccgagctgcaccgcccaggccctcctcccctccgagcagccccactcctgcccccaggggcaagagggctaccccctcctcctcctcccctgccccctccactccctcctcgtctcagagaggacgctgaagagcaggaaaccacctgccccatttgcctgggggagatacagaatgccaagacgctggagaagtgcaggcactccttctgtgagggctgcatcacacgggccctgcaggtgaaaaaggcctgccccatgtgcggccgattctacgggcagcttgtgggcaaccagccccagaacggacggatgctggtctccaaggatgccaccctcctgctgcccagctacgagaagtatggcaccattgtcatccaatatgtcttcccgcctGGCGTCCAGGGGGTAAGAGATCACAGTTCCTACCCGTTGGCTTCCTCCAAGTTCACTGTCTCTTGACTTAAGAAAAAAAGGCAATGTAGAGAGTATGTTGTTAG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37338 GAT GCT GGT CTC CAA GGA TG Wild type Forward A
37339 TGA TCT CTT ACC CCC TGG AC Common A
37340 GGT TCA GAA GGA GCC TGA AG Mutant Forward A
37341 Fluorophore-1 AGT CCC TTT CAC CCT GGG Quencher-1 MUT Probe
37342 Fluorophore-2 CTC CTG CTG CCC AGC TAC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37338 0.40 uM
37339 0.40 uM
37340 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.