Protocol 30997: Probe Assay - Washc4<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

>chr10:83017380+83017469 90bp GCTGGATAGCTAGAGCTTTGGT CATGGTGAGTACGCGTTACAAG

Mutant=  96 bp

Wild Type = 90 bp

Sequence

Wt Sequence:gctggatagctagagctttggttgtgttacagcatccagtctaaatatccCGacactgacaaagcagtcttgtaacgcgtactcaccatg

 

Mutant Sequence: gctggatagctagagctttggttgtgttacagcatccagtctaaatatccCGaggttgtgttccttttttaaggtgtgtgcttacagataattgag

258 bp deletion beginning at Chromosome 10 positive strand position 83,554,686 bp GACACTGACAAAGCAGTCTT, and ending after AGGATGTTCAGACACTGCTG at 83,554,943 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35931 GCT GGA TAG CTA GAG CTT TGG T Common A
35932 CAT GGT GAG TAC GCG TTA CAA G Wild type Reverse A
35933 CTC AAT TAT CTG TAA GCA CAC ACC Mutant Reverse A
35934 Fluorophore-1 AAT ATC CCG AGG TTG TGT TCC Quencher-1 MUT Probe
35935 Fluorophore-2 AAT ATC CCG ACA CTG ACA AAG C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35931 0.40 uM
35932 0.40 uM
35933 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.