Stock No: 008223
Protocol 8872: Restriction Enzyme Digest Assay - Fasl<gld>
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-Faslgld/J
000784 C3H/HeJ-Faslgld/J
002932 CPt.C3-Faslgld/J
008223 NOD.C3(B6)-Faslgld /LwnJ
4 strains use this protocol