Protocol 45306: Probe Assay - Pom121l2<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

>chr13:21981553+21981639 87bp CTCTCAGCATGGGCAGCTA CAGGCTTCTCCCCTGAGTCT

Mutant=  81 bp

Wild Type = 87 bp

Sequence

Wt Sequence: ctctcagcatgggcagctacctgggCAAAGCGGGGTCCTCGCCGCGGTCCCCAGCACAGGGGCGCGCAGACTCAGGGGAGAAGCCTG

Mut Sequence: CTCTCAGCATGGGCAGCTACCTGGgcATCACACTTACAAGAAATAGCCCACTCTCTCTGTCACACTGCAACAGAGTCTTGA

 

A 2881 bp deletion beginning at Chromosome 13 position 21,981,578 bp and ending after 21,984,458 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65247 CTC TCA GCA TGG GCA GCT A Common A
65248 CAG GCT TCT CCC CTG AGT CT Wild type Reverse A
65249 TCA AGA CTC TGT TGC AGT GTG A Mutant Reverse A
65250 Fluorophore-1 TGG GCA TCA CAC TTA CAA GAA ATA Quencher-1 MUT Probe
65251 Fluorophore-2 TGG GCA AAG CGG GGT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65247 0.40 uM
65248 0.40 uM
65249 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.