Protocol 40820: Probe Assay - Kcnn3<em1.1Lutzy>
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

>chr3:89651898+89651981 84bp CTCAAAGTCTTATACCATTCCCTTG CACACAGTTAGGAGGTAAAACACA

Mut= 114 bp

Wt= 84 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence:

acaagctttatggcatgtcttgaccaattagagactcaaagtcttataccattcccttgaaatgccaacactaagcctggggcacaccaagtcttgtgttttacctcctaactgtgtgttttctctcctgggaccctctgacgtcctctggcagGGTGCAGGCTGCAC

Mutant Sequence:

GGGCGCAAGCAGGTTTTGACAAGCTTTATGGCATGTCTTGACCAATTAGAGACTCAAAGTCTTATACCATTCCCTTGAAATGCCAACACTAAGCCTATAACTTCGTATAGCATACATTATACGAAGTTAtCACACCAAGTCTTGTGTTTTACCTCCTAACTGTGTGTTTTCTCTCCTGGGACCCTCTGACGTCCTCTGGCAGGGTGCAGGCTGCACTGCCCTTGTGG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
56863 CTC AAA GTC TTA TAC CAT TCC CTT G Forward A
56864 CAC ACA GTT AGG AGG TAA AAC ACA Reverse A
56865 Fluorophore-1 AAG CCT GGG GCA CAC C Quencher-1 WT Probe
56866 Fluorophore-2 CAC TAA GCC TAT AAC TTC GTA TAG CAT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
56863 0.40 uM
56864 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.