Protocol 45576: Probe Assay - Lrch3<em1(IMPC)J> Alt 1
Version 2.0

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

>chr16:32950183+32950302 120bp CAGTTTCCACTAGGAATTCTGGA TCGTGACAGGTCTGAAAGAAAA

Mutant=  80 bp

Wild Type = 120 bp

Sequence

Wt Sequence: cagtttccactaggaattctggagaaatcaagtaattatgaagcatactaActaatttcagtaaaacaagtttcaaaggatggttttaagcttttatattttctttcagACCTGTCACGA

Mut Sequence: cagtttccactaggaattctggagaaatcaagtaattatgaagcatactaAGcctggtatgcacaagacactggttcagc

A 476 bp deletion beginning at Chromosome 16 position 32,950,234 bp and ending after 32,950,709 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65582 CAG TTT CCA CTA GGA ATT CTG GA Common A
65583 TCG TGA CAG GTC TGA AAG AAA A Wild type Reverse A
65584 Fluorophore-1 AGT TTC AAA GGA TGG TTT TAA GCT T Quencher-1 WT Probe
65932 GCT GAA CCA GTG TCT TGT GC Mutant Reverse A
65933 Fluorophore-2 AGT AAT TAT GAA GCA TAC TAA GCC TGG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65582 0.40 uM
65583 0.40 uM
65932 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.