Stock No: 005124
Protocol 4111: Restriction Enzyme Digest Assay - Mpl<hlb219>
Version 2.0

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 =196 bp and 190 bp (cut)
Heterozygote = 386 bp and 196 bp and 190 bp
Wild type =386 bp (not cut)

Sequence

GCTGGCCTTGGGCACAGAGCCCCTGAAC(t/c)GCTTCTCCCAAACATTTGAGGACCT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9746 GCC AAG GTG AGG TGG ATA GA Forward
9747 TCC TCT GAT AGG GCC AAA GA Reverse

Reaction A

Component Final Concentration
ddH2O
AB PCR BufferII 1.00
MgCl2 2.00
dNTP 0.20
9746 0.10
9747 0.10
DNA Loading Dye 0.69
Taq DNA Polymerase 0.03
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 62.0 --
4 72.0 --
5 -- Go to step 2-4, 35 times
6 72.0 --
7 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
NEB Buffer 3 1.00
AciI 0.83
DNA

Cycling

Step Temp °C Time Note
1 37.0 --
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.