Protocol 8872: Restriction Enzyme Digest Assay - Fasl<gld>

B6Smn.C3-Fasl^gld/J

Stock No: 001021

Version 3.0

Notes

Incubate 8 of PCR product with 4 µl restriction enzyme master mix for a minimum of 4 hours at 37 degrees 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 = 108 bp
Heterozygote = 108 bp and 88 bp and 20 bp
Wild type = 88 bp and 20 bp

Add DNA Loading Dye* to digested products and separate on a 3% agarose gel.**

JAX Protocol

Protocol Primers

Primer	5' Label	Sequence 5' → 3'	3' Label	Primer Type	Reaction	Note
oIMR1110		CAA GAC AAT ATT CCT GGT GCC		Reverse
oIMR1163		CAA TTT TGA GGA ATC TAA GGC C		Forward

Reaction A

Component	Final Concentration
ddH2O
AB PCR BufferII	1.00 X
MgCl2	2.00 mM
dNTP	0.20 mM
oIMR1163	1.00 uM
oIMR1110	1.00 uM
DNA Loading Dye	0.69
Taq DNA Polymerase	0.03 U/ul
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	55.0	--
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
Rest Enzyme Buffer	1.00
ddH2O
Stu I	0.83 U/ul
PCR product

Cycling

Step	Temp °C	Time	Note
1	37.0	--	2-6 hours
2	10.0	--	hold

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
001021	B6Smn.C3-Fasl^gld/J
000784	C3H/HeJ-Fasl^gld/J
002932	CPt.C3-Fasl^gld/J
008223	NOD.C3(B6)-Fasl^gld /LwnJ
4 strains use this protocol