Stock No: 028892
Protocol 21569: Probe Assay - Mbl1<tm1.1(MBL2)Glst> 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

Mutant=  113 bp

Wild Type = 110 bp

Sequence

Wt Sequence: gttggaagccctggaagtccaggaccaaaaggccaaaagggggaccatggagacaatagaggtaagagaatgccttagaatgattagcttgagtgcaggtatgtgtccga

 

Mutant Sequence: ATCTGGCCGTCTGTGAGTTCCCTATCTGAtagtaagtcgacgaagttcctatactttctagagaataggaacttcctcgagaccgtacggaaacgagtgcctccatat

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
32743 GTT GGA AGC CCT GGA AGT C Wild type Forward A
32744 TCG GAC ACA TAC CTG CAC TC Wild type Reverse A
32745 Fluorophore-1 GGG GGA CCA TGG AGA CA Quencher-1 WT Probe
32746 ATC TGG CCG TCT GTG AGT TC MUT Probe A
32747 ATA TGG AGG CAC TCG TTT CC Mutant Reverse A
32749 Fluorophore-2 AGA GAA TAG GAA CTT CCT CGA GAC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
32743 0.40 uM
32744 0.40 uM
32746 0.40 uM
32747 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.