Stock No: 008185
Protocol 30543: Probe Assay - Kras<tm3Tyj> 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=  90 bp

Wild Type = 101 bp

Sequence

Wt Sequence: ccagtttaatatctgcctgggttacagagcaggtgcaaggtcagcctgcacagcttagtgagaccctgtctagAGtgaagaatgaaggaggggctgtggctatagcccattggtgaagtgcttgtactttgggattccattacatctttacatttaaaaaatggcaggatactttttaaaaatttaagtgataataccattacattt

 

Mutant Sequence: agcaggtgcaaggtcagcctgcACAGCTTAGTGAGACCCTGTCTAGAactagtggatcccccgggctgcagGAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34854 GGT CAG CCT GCA CAG CTT AG Common A
34855 TGG AAT CCC AAA GTA CAA GCA Wild type Reverse A
34856 Fluorophore-1 CCC TGT CTA GAA CTA GTG GAT CCC Quencher-1 MUT Probe
34857 Fluorophore-2 CCT GTC TAG AGT GAA GAA TGA AGG AG Quencher-2 WT Probe
oIMR6592 GAA AAG CGC CTC CCC TAC Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34854 0.40 uM
34855 0.40 uM
oIMR6592 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.