Stock No: 003548
Protocol 176: Restriction Enzyme Digest Assay - Ins2<Akita>
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
006860 B6.Cg-Ins2Akita Bdkrb2tm1Jfh/SmiJ
006580 B6.Cg-Ins2Akita Ldlrtm1Her/J
004369 B6.Cg-Rag1tm1Mom Ins2Akita/J
003548 C57BL/6-Ins2Akita/J
007562 D2.B6-Ins2Akita/MatbJ
006867 FVB.B6-Ins2Akita/MlnJ
008659 NOD.Cg-Rag1tm1Mom Ins2Akita Prf1tm1Sdz/SzJ
014568 NOD.Cg-Rag1tm1Mom Ins2Akita Il2rgtm1Wjl/SzJ
8 strains use this protocol