Protocol 31318: Probe Assay - Lrrc55<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mutant=  80 bp

Wild Type = 128 bp

>chr2:85195944-85196071 128bp CCCTACAGATCGGGGGTAAC TTCTGCCTGGTGACCTGT

Sequence

Wt Sequence:

 

Mutant Sequence:

702 bp deletion beginning at Chromosome 2 negative strand position 85,196,683 bp and ending after 85,195,982 bp (GRCm38/mm10). This mutation deletes 702 bp from ENSMUSE00000643986 (exon 1) including the ATG start site but retains the last 3 bp (CGG) in the exon

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36610 AGC AGC ATC CCT GTA ACT TGA Mutant Forward A
36611 TTC TGC CTG GTG ACC TGT Common A
36612 CCC TAC AGA TCG GGG GTA AC Wild type Forward A
36613 Fluorophore-1 AGG GAC ACC GCG GGT A Quencher-1 MUT Probe
36614 Fluorophore-2 CAT GGA GCC CTT GCT GAA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36610 0.40 uM
36611 0.40 uM
36612 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.