Stock No: 012810
Protocol 7631: Restriction Enzyme Digest Assay - Enpp1<asj>
Version 1.0

Notes

Add 4 ul amplified PCR product to 8 ul RE mix and incubate @65C x 4hours.

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 = ~150 bp

Wild type = 300 bp

uncut = 300 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13013 TGA TCT GCA TCC TGG GAT AA Forward
13014 TAA GGA AAG ACC AAT TGC AGA Reverse

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.00 X
MgCl2 2.00 mM
dNTPS-kapa 0.20 mM
13013 0.50 uM
13014 0.50 uM
10x Loading Dye 0.50 mM
Kapa 2G HS taq polym 0.01 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -1.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles
6 94.0 --
7 50.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28cycles
10 72.0 --
11 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 X
NEB buffer 4 1.50
BSA 0.15
TaqaI 30.00

Cycling

Step Temp °C Time Note
1 65.0 --
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.