Protocol 176: Restriction Enzyme Digest Assay - Ins2<Akita>

C57BL/6-Ins2^Akita/J

Stock No: 003548

Version 1.1

Notes

Incubate 8 µl restriction digest with 4 µl PCR product for a minimum of 3 hours at 37 C prior to running on gel.

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 = 280 bp
Heterozygote = 140 bp and 280 bp
Wild type = 140 bp

Separate digested products on a 3.0% agarose gel.**

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
oIMR1093		TGC TGA TGC CCT GGC CTG CT		Forward
oIMR1094		TGG TCC CAC ATA TGC ACA TG		Reverse

Reaction A

Component	Final Concentration
ddH2O
AB PCR BufferII	1.00 X
MgCl2	2.00 mM
dNTP	0.20 mM
oIMR1093	0.80 uM
oIMR1094	0.80 uM
DNA Loading Dye	0.69 mM
Taq DNA Polymerase	0.01 U/ul
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	64.0	--	*-0.5 C per cycle
4	72.0	--	repeat steps 2-4 for 12 cycles
5	94.0	--
6	58.0	--
7	72.0	--	repeat steps 5-7 for 25 cycles
8	72.0	--
9	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
ddH2O
BSA	mg/ml
Fnu4HI

Strains Using This Protocol

Stock Number	Strain Name
012307	B6.129-Apoe^tm3(APOE*4)Mae Ldlr^tm1(LDLR)Mae/J
006860	B6.Cg-Ins2^Akita Bdkrb2^tm1Jfh/SmiJ
006580	B6.Cg-Ins2^Akita Ldlr^tm1Her/J
004369	B6.Cg-Rag1^tm1Mom Ins2^Akita/J
003548	C57BL/6-Ins2^Akita/J
007562	D2.B6-Ins2^Akita/MatbJ
006867	FVB.B6-Ins2^Akita/MlnJ
008659	NOD.Cg-Rag1^tm1Mom Ins2^Akita Prf1^tm1Sdz/SzJ
014568	NOD.Cg-Rag1^tm1Mom Ins2^Akita Il2rg^tm1Wjl/SzJ
9 strains use this protocol