Stock No: 004125
Protocol 32758: Probe Assay - Abcb11<tm1Wng> Probe Alternate1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

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=  77 bp

Wild Type = 96 bp

Sequence

Wt Sequence:

 

actttctaaagaatgttcttcccttgtcagATTTTAAATAACCTCAGCATGGTCATTAAGCCAGGGGAAACGACGGCGTTCGTGGGATccagtGGGGCAGG

GAAGAGCACAGCACTACAGCTCATTCAGAGATTCTATGACCCCTGCGAAGGCATGgtgcgtatcttctagaaacttctcaacatggaggggtaagg

 

Mutant Sequence: 

 

TTTAAATAACCTCAGCATGGTCATTAAGCCAGGGGAAACGACGGCGTTCGTGGGATCCcccgatctatagatcatgagtgggaggaatgagctggcc

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
29884 TCA TTA AGC CAG GGG AAA CG Common A
29885 CGC AGG GGT CAT AGA ATC TC Wild type Reverse A
29887 Fluorophore-1 TGA GCT GTA GTG CTG TGC TCT Quencher-1 WT Probe
39907 Fluorophore-2 CGT GGG ATC CCC CGA TCT ATA G Quencher-2 MUT Probe
oIMR1653 GGC CAG CTC ATT CCT CCC ACT CAT G Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
29884 0.40 uM
29885 0.40 uM
oIMR1653 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.