Protocol 38058: End Point Analysis Assay - Kif1a<em13(indel)Lutzy>
Version 1.0

Notes

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 = T
Heterozygote = -/T
Wild type = -

Mutation is a T insertion.

Sequence

WT sequence showing the {insertion}:

ACTACCAACCAACTCCACAGAAATGGCCGGGGCCTCTGTGAAGGTGGCGGTGCGTGTCCGCCCCTT{t}CAACTCCCGGGAAATGAGCCGAGACTCCAAGTGCATCATTCAGATGTCTGGAAGCACCACCA

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52892 GGG GCC TCT GTG AAG GTG Forward A
52893 TTG GAG TCT CGG CTC ATT TC Reverse A
52894 Fluorophore-1 CGC CCC TTC AAC TCC C Quencher-1 WT Probe
52895 Fluorophore-2 CGC CCC TTT CAA CTC CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52892 0.40 uM
52893 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.