Protocol 30557: Probe Assay - Styxl1<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

>chr5:136244769-136244859 91bp TTACTCACCTGCCCACCAGT TCGTTTTGATCGGACATCTG

Mutant=  90 bp

Wild Type = 91 bp

Sequence

Wt Sequence: ttactcacctgcccaccagtgttctcatgtcccttcACgtgtccagttctgagttcaacccttttgttccccagATGTCCGATCAAAACGA

 

Mutant Sequence: ttactcacctgcccaccagtgttctcatgtcccttcATgcatctgctctctgcgtcctccgggatgcctgtgagcttggtgctgtgtcta

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34882 TTA CTC ACC TGC CCA CCA GT Common A
34883 TCG TTT TGA TCG GAC ATC TG Wild type Reverse A
34884 TAG ACA CAG CAC CAA GCT CA Mutant Reverse A
34885 Fluorophore-1 TGT CCA GTT CTG AGT TCA ACC C Quencher-1 WT Probe
34886 Fluorophore-2 TGC GTC CTC CGG GAT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34882 0.40 uM
34883 0.40 uM
34884 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.