Protocol 31360: Probe Assay - Scrn3<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

>chr2:73318403+73318497 95bp CACTTTTGGGCATGGACCT GAAAATGGATGTGGGTTGGT

Mutant=  90 bp

Wild Type = 95 bp

Sequence

Wt Sequence: CACTTTTGGGCATGGACCTTGTCAGgtaacagttgtgctgctttcccactgtagatgatgtgggaaatGGtaaataccaacccacatccattttc

 

Mutant Sequence: agtgctcttaaccactgaaccatctctccagcccctgcatgagttttattattgaaaatttaaTGtaaataccaacccacatccattttc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36697 CAC TTT TGG GCA TGG ACC T Wild type Forward A
36698 GAA AAT GGA TGT GGG TTG GT Common A
36699 AGT GCT CTT AAC CAC TGA ACC AT Mutant Forward A
36700 Fluorophore-1 CAG GTA ACA GTT GTG CTG CTT T Quencher-1 WT Probe
36701 Fluorophore-2 CCA GCC CCT GCA TGA GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36697 0.40 uM
36698 0.40 uM
36699 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.