Stock No: 028377
Protocol 17987: End Point Analysis Assay - Engase<em2Lutzy>-EP1
Version 4.0

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 = ----/----
Heterozygote = GTGT/----
Wild type = GTGT/GTGT

Sequence

CTAGATATTATGACAAGGATACTACCAGACCTATCAGCTTTTACCTGTCGACGCTGGAGGA
GCTCCTGGCCTGGACCCCGCTCATGGAGGATGGCTTCAATGTAGCCCTGGAACCCCTGGT[gtgt]
CGCCGGCCTCCACTCAGCAGCCCCAGGCCCCGGACCCTGCTGTGCCACGACATGATGGGTGGTT
ACCTGGAGGACAG
 
Mutation is a 4 bp deletion.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25525 GCT CAT GGA GGA TGG CTT C Forward A
25526 CAT GTC GTG GCA CAG CAG Reverse A
25527 Fluorophore-1 CCT GGT GTG TCG CCG G Quencher-1 Internal Positive Control Forward A
25528 Fluorophore-2 CCC TGG TCG CCG GC Quencher-2 Internal Positive Control Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa Probe Fast QPCR 1.00 X
25525 0.40 uM
25526 0.40 uM
25527 0.40 uM
25528 0.40 uM
Tg Probe 0.15 uM
IC 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.