Protocol 45414: Probe Assay - Wdr38<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

>chr2:38999897+38999987 91bp AGGCCTTGTGGTTGGAACT AAGACTTCACTGGGCCTGAG

Mutant=  93 bp

Wild Type = 91 bp

Sequence

Wt Sequence: aggccttgtggttggaactacagataacaaatctggacCtggagggcaggacagtgccttgtgttctctgcctcagGCCCAGTGAAGTCTT

Mut Sequence:  aggccttgtggttggaactacagataacaaatctggacCCtcttagtgcctcccctactctctccccacacccaatcagatgcaggttccagc

 

A 381 bp deletion beginning at Chromosome 2 position 38,999,936 bp and ending after 39,000,316 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65551 AGG CCT TGT GGT TGG AAC T Common A
65552 AAG ACT TCA CTG GGC CTG AG Wild type Reverse A
65553 GCT GGA ACC TGC ATC TGA TT Mutant Reverse A
65554 Fluorophore-1 TCT GGA CCC TCT TAG TGC CTC Quencher-1 MUT Probe
65555 Fluorophore-2 ACA GTG CCT TGT GTT CTC TGC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65551 0.40 uM
65552 0.40 uM
65553 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.