Protocol 31114: Probe Assay - Cep192<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

>chr18:67967120+67967227 108bp TTGCTTGGTATCTGTCTTCTGC CATGGTTCCCAAGACAAAGG

Mutant=  116 bp

Wild Type = 108 bp

Sequence

Wt Sequence: ttgcttggtatctgtcttctgcttttatcctgtggtaagcagtgttttggtttgtcacGGgcttttgtagagctttcttgatgaatttcctttgtcttgggaaccatg

 

Mutant Sequence: catgagaggcagggtgatggtgattattctctttggagctggggaactctgtgccacagaagcccaCGgcttttgtagagctttcttgatgaatttcctttgtcttgggaaccatg

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36160 TTG CTT GGT ATC TGT CTT CTG C Wild type Forward A
36161 CAT GGT TCC CAA GAC AAA GG Common A
36162 CAT GAG AGG CAG GGT GAT G Mutant Forward A
36163 Fluorophore-1 CCT GTG GTA AGC AGT GTT TTG G Quencher-1 WT Probe
36164 Fluorophore-2 CTC TTT GGA GCT GGG GAA C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36160 0.40 uM
36161 0.40 uM
36162 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.