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Restriction Digest of Plasmid DNA

Introduction

Restriction enzyme digestion takes advantage of naturally occurring enzymes that cleave DNA at specific

sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of

recognition sequences. For a list of many commonly used restriction enzymes, visit NEB.

Restriction enzyme digestion is commonly used in molecular cloning techniques, such as PCR or restriction cloning.

It is also used to quickly check the identity of a plasmid by diagnostic digest.

Last Upload: Oct. 11th, 2016

You may

Recovering Plasmid DNA

from Bacterial Culture

Purifying DNA from an

Agarose Gel

DNA Ligation

Equipment

•Electrophoresis chamber

•Pipetman

Reagents

•Liquid DNA aliquot of your plasmid of interest (see below for

recommend amounts)

•Appropriate restriction enzyme (see manufacturer's

instructions for proper ammount)

•Approrpriate restriction digest buffer (see manufacturer's

instructions)

•Gel loading dye

•Electrophoresis buffer

•Pipet tips

Procedure

restriction enzymes to digest your plasmid.

*Pro-Tip* To determine which restriction enzymes will cut your DNA sequence (and where they will cut), use a sequence

analysis program such as Addgene's Sequence Analyzer.

ine an appropriate reaction buffer by reading the instructions for your enzyme.

*Pro-Tip* If you are conducting a double digest (digesting with two enzymes at the same time), you will need to determine the

best buffer that works for both of your enzymes. Most companies will have a compatibility chart, such as the double digest

finder tool from NEB.

a 1.5mL tube combine the following:

◦

◦

◦

◦

◦

DNA

Restriction Enzyme(s)

Buffer

BSA (if recommended by manufacturer)

dH

2

O up to total volume

*Pro-Tip* The amount of DNA that you cut depends

on your application. A diagnostic digest typically

involves ∼500 ng of DNA, while molecular cloning

often requires 1 µg of DNA. The total reaction

volume usually varies from 10-50 µL depending on

application and is largely determined by the volume

of DNA to be cut.

*Pro-Tip* A typical restriction digestion reaction

could look like this:

◦1 µg DNA

◦1 µL of each Restriction Enzyme

◦3 µL 10x Buffer

◦3 µL 10x BSA (if recommended)

◦x µL dH

2

O (to bring total volume to 30µL)

*Pro-Tip* The amount of restriction enzyme you use for a given digestion will depend on the amount of DNA you want to cut.

By definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour. Using this ratio, you can calculate the

minimal amount of enzyme for your reaction. However, keep in mind that restriction enzyme activity is determined under ideal

conditions with very clean DNA, so using a little more enzyme is advisable. Reactions are often performed with 0.2-0.5 µL of

enzyme because it is difficult to pipette less volume than this; 0.2-0.5 µL will likely be more enzyme than you will need, but

that's okay because a little more enzyme is usually better.

gently by pipetting.

te tube at appropriate temperature (usually 37 °C) for 1 hour. Always follow the manufacturer’s instructions.

*Pro-Tip* Depending on the application and the amount of DNA in the reaction, incubation time can range from 45 mins to

overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg of DNA used for cloning, it is

recommended that you digest for at least 4 hours.

*Pro-Tip* If you will be using the digested DNA for another application (such as a digestion with another enzyme in a different

buffer), but will not be gel purifying it, you may need to inactivate the enzyme(s) following the digestion reaction. This may

involve incubating the reaction at 70 °C for 15 mins, or purifying the DNA via a purification kit, such as a QIAGEN DNA cleanup

kit. See the enzyme manufacturer's instructions for more details.

visualize the results of your digest, conduct gel electrophoresis.

Tips and FAQ

•Restriction enzymes MUST be placed in an ice bucket immediately after removal from the -20 °C freezer because heat can cause the

enzymes to denature and lose their function.

•If you are having difficulty finding an

enzyme that cuts your vector's multiple

cloning site (MCS), but does not cut your

insert, you could try using two different

enzymes that have compatible sticky ends.

See NEB's compatible cohesive ends

chart.

•If you cannot find compatible sticky ends,

you will need to fill in the overhangs and

conduct a blunt end ligation. Use T4 DNA

Polymerase or Klenow DNA Polymerase I

for 3′ overhang removal and 5′ overhang fill-

in.

•If you are using blunt ends or a single

enzyme to cut the vector, you will need to

use a phosphatase to prevent re-

circularization of the vector if you are

cloning in an insert. CIP (calf alkaline

phosphatase) or SAP (shrimp alkaline

phosphatase) are commonly used. Follow

the manufacturer’s instructions.

•If your enzyme did not cut, check to make sure that it isn't