Stock No: 034602
Protocol 39692: Probe Assay - Kcnn3<em1/2lutzy>
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

>chr3:89651915+89652004 90bp TTCCCTTGAAATGCCAACAC TCAGAGGGTCCCAGGAGAGA

Mut= 115 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence:

ttagagactcaaagtcttataccattcccttgaaatgccaacactaagcctggggcacaccaagtcttgtgttttacctcctaactgtgtgttttctctcctgggaccctctgacgtcctc

Mutant Sequence:

taccattcccttgaaatgccaacactaagcctATAACTTCGTATAGCATACATTATACGAAGTTATATACTCTGTATCCTGTTTTCAGTTCATAGCCCATCTGTAGGGCGCAGTAGTCCAGGGTTTCCTTGATG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53820 TGG ACT ACT GCG CCC TAC AG Mutant Reverse A
53822 Fluorophore-1 ATC CTG TTT TCA GTT CAT AGC CCA Quencher-1 MUT Probe
54406 TTC CCT TGA AAT GCC AAC AC Common A
54407 TCA GAG GGT CCC AGG AGA GA Wild type Reverse A
54408 Fluorophore-2 AAG CCT GGG GCA CAC CA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53820 0.40 uM
54406 0.40 uM
54407 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.