Stock No: 010825
Protocol 150: Restriction Enzyme Digest Assay - Ptpn6<me>
Version 1.1

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 = 318 bp and 23 bp
Heterozygote = 318 bp, 164 bp, 154 bp, and 23 bp
Wild type = 164 bp, 154 bp, and 23 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
oIMR4155 TGC CTA TGT GCT TTT GCA TC Common
oIMR4156 CCT GGT CTG GAT GTC CAC TT Common

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00 X
MgCl2 2.00 mM
dNTP 0.20 mM
oIMR4155 1.00 uM
oIMR4156 1.00 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 66.0 --
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
AB PCR BufferII X
ddH2O
BSA mg/ml
TaqaI

Strains Using This Protocol

Stock Number Strain Name
000225 C3FeLe.B6 a/a-Ptpn6me/J
000810 C57BL/6J-Ptpn6me/J
010825 C57BL/6J-Ptpn6me/SzJ
3 strains use this protocol