Stock No: 011121
Protocol 21811: Probe Assay - Axl<tm1Grl> Probe Alternate 1
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=  109 bp

Wild Type = 100 bp

Sequence

Wt Sequence: tggagacaaatgtagagacaaggggaaaggatgaacaagagttgagtcttaggagttctagATgtaggtgtccctcctgatgtcgcctctggacttccta

 

Mutant Sequence: TGGAGACAAATGTAGAGACAAGGGGAAAGGATGAACAAGAGTTGAGTCTTAGGAGTTCTAGAgtcagcttctgatggaattagaacttg

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32955 TGG AGA CAA ATG TAG AGA CAA GG Common A
32956 TAG GAA GTC CAG AGG CGA CA Wild type Reverse A
32958 Fluorophore-1 TTC TAG ATG TAG GTG TCC CTC CTG Quencher-1
33299 Fluorophore-2 AGT TCT AGA GTC AGC TTC TGA TGG A Quencher-2
9715 TTT GCC AAG TTC TAA TTC CAT C Mutant A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32955 0.40 uM
32956 0.40 uM
9715 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 4.0 -- 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.