Protocol 36645: Probe Assay - Pdxp<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

>chr15:78919572+78919677 106bp CCATAGCCCACAAGAAAGGA CCTACAGGGAAAGGCCTCAG

Mut = 114 bp

Wt = 106 bp

Fam = Mut

Hex = Wt

Sequence

MUT Sequence:

gcccgaagacaacatctctctcagcctctgtttttctcctagcaaccatctggcctacgtttttAAcattttgcccattgagcttttagcatctctgaggcctttccctgtagg

This mutation is a 5911 bp deletion beginning at Chromosome 15 position 78,913,713 bp and ending after 78,919,623 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49303 CCT ACA GGG AAA GGC CTC AG Common A
49304 CCA TAG CCC ACA AGA AAG GA Wild type Forward A
49305 GCC CGA AGA CAA CAT CTC TC Mutant Forward A
49306 Fluorophore-1 CCC ATC TCT GTG TCC TCA GCC Quencher-1 WT Probe
49307 Fluorophore-2 AAC CAT CTG GCC TAC GTT TTT AAC ATT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49303 0.40 uM
49304 0.40 uM
49305 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.