Protocol 550: Restriction Enzyme Digest Assay - Apob<tm2Sgy>

B6;129-Apob^tm2Sgy Apoe^tm1Unc/J

Stock No: 002879

Version 1.1

Notes

This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).

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 = 2500 bp and 300 bp
Heterozygotes = 2800 bp and 2500 bp and 300 bp
Wild type = 2800 bp (uncut)

Separated by gel electrophoresis on a 1.5% agarose gel.**

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
oIMR1745		TCC TGA GCT GCT GTA GTT TTT C		Common
oIMR1776		CCG CAT GAA GAC TGA CTC TG		Common

Reaction A

Component	Final Concentration
ddH2O
AB PCR BufferII	1.00 X
MgCl2	2.50 mM
dNTP	0.20 mM
oIMR1776	0.50 uM
oIMR1745	0.50 uM
Taq DNA Polymerase +	0.02 U/ul
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	65.0	--
4	72.0	--	repeat steps 2-4 for 35 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.

Reaction B

Component	Final Concentration
AB PCR BufferII	X
Hind III

Strains Using This Protocol

Stock Number	Strain Name
002879	B6;129-Apob^tm2Sgy Apoe^tm1Unc/J
003000	B6;129S-Ldlr^tm1Her Apob^tm2Sgy/J
002877	B6;129S7-Apob^tm2Sgy/J
004192	STOCK Mttp^tm2Sgy Ldlr^tm1Her Apob^tm2Sgy Tg(Mx1-cre)1Cgn/J
4 strains use this protocol