Protocol 2149: End Point Analysis Assay - Acd<acd>

DW/J-Acd^acd/J

Stock No: 001595

Version 1.1

Notes

End point analysis is done using Amplifluor SNPS Genotyping System from Millipore. End point analysis protocols are run on an ABI 7000, 7500, or the Roche LightCycler 480

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 = green
Wild type = red

Followed by End Point Analysis on ABI 7000

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
oIMR4221		GAA GGT GAC CAA GTT CAT GCT TCG CCC AAG TGA GCC TTA CCA T		Mutant
oIMR4222		GAA GGT CGG AGT CAA CGG ATT GCC CAA GTG AGC CTT ACC AC		Wild type
oIMR4223		CTC AGG ACC ATG CGG TGA		Common

Reaction A

Component	Final Concentration
ddH2O
Ampliflour SNP FAM	0.83 X
SNP JOE	0.83 X
Primer Mix	0.83 X
Rxn Mix S+	0.83 X
dNTP	0.17 mM
Titanium Taq Pol.	0.04 U/ul
DNA

Cycling

Step	Temp °C	Time	Note
1	95.0	--
2	94.0	--
3	55.0	--
4	72.0	--	repeat steps 2-4 for 40 cycles
5	72.0	--
6	10.0	--	hold

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.