Protocol 45308: Probe Assay - Ppp1r36<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

>chr12:76427586+76427680 95bp CCCCTCAAGCTCTCATTGC TCACTGCTCTGTCCCTCAAA

Mutant=  84 bp

Wild Type = 95 bp

Sequence

Wt Sequence: cccctcaagctctcattgcaagccaattcCctgtgtatctcaggatacactagatacagctgaattaaaatattctttgagggacagagcagtga

Mut Sequence: cccctcaagctctcattgcaagccaattcCCtgggcttgtgcctctgtaatggcactgtgtgattctgggagttgttcatacca

A 10249 bp deletion beginning at Chromosome 12 position 76,427,616 bp and ending after 76,437,864 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65257 CCC CTC AAG CTC TCA TTG C Common A
65258 TCA CTG CTC TGT CCC TCA AA Wild type Reverse A
65259 TGG TAT GAA CAA CTC CCA GAA Mutant Reverse A
65260 Fluorophore-1 CAA TTC CCT GGG CTT GTG Quencher-1 MUT Probe
65261 Fluorophore-2 TGT GTA TCT CAG GAT ACA CTA GAT ACA GCT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65257 0.40 uM
65258 0.40 uM
65259 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.