Protocol 5973: Restriction Enzyme Digest Assay - Kit-<W-v>

C57BL/6J-Kit^W-v/J

Stock No: 000049

Version 2.0

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 = 192 bp and 76bp
Heterozygote = 268 bp and 192 bp and 76 bp
Wild type= 268 bp

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
oIMR9392		GCC CTA ATG TCG GAA CTG AA		Forward
oIMR9393		GTC TGA AGG CTC CCA GCA TA		Reverse

Reaction A

Component	Final Concentration
ddH2O
AB PCR BufferII	1.00 X
MgCl2	2.00 mM
dNTP	0.20 mM
oIMR9392	1.00
oIMR9393	1.00
Taq DNA Polymerase	0.03 U/ul
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	60.0	--
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
ddH2O	0.00
NEB Buffer 3	1.50 X
NsiI

Cycling

Step	Temp °C	Time	Note
1	37.0	--

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number	Strain Name
016588	A.B6-Kit^W-v/BeiMmjax
000049	C57BL/6J-Kit^W-v/J
2 strains use this protocol