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Double digests

Double digests

Generally, a vector and an insert DNA to be cloned are produced by cleaving with two different restriction endonucleases, thus generating two different restriction ends. This strategy prevents the vector from being ligated without an insert, resulting in great reduction in self-ligation and increase in cloning efficiency. Over 95% of restriction enzymes are 100% active in FastCut buffer (former name: EZ-One™ buffer), making double digestion simple. If you are using an enzyme that is not supplied with FastCut buffer (former name: EZ-One™ buffer), the Performance Chart for Restriction Enzymes rates the percentage activity of each restriction endonuclease in the four standard NEBuffers.

 

Setting up a Double Digestion

l  Simultaneous DNA cleavage with two restriction endonucleases in a common restriction buffer saves time and efforts.

l  Double digests with Enzynomics’ restriction enzymes can be set up in FastCut buffer (former name: EZ-One™ buffer). Otherwise, choose an EzBuffer that results in the most activity for both enzymes. If star activity is a concern, consider using one of our EZ-CleanCut™ enzymes.


[Example] For the performing double digestion reaction using Not I and Pst I, simply select a EzBuffer III because both enzymes are 100% active on EzBuffer III. Alternatively, FastCut buffer (former name: EZ-One™ buffer) can be used because both enzymes are compatible with FastCut buffer (former name: EZ-One™ buffer) system.

Enzyme

Cat.#

Supplied EzBuffer

% Activity in EzBuffers

I

II

III

IV

Not I

R001

III

0

50

100

0

Pst I

R019

III

100

100

100

75

 

l  Set up reaction according to standard protocol. The final concentration of glycerol in any reaction should be less than 5% to minimize the possibility of star activity. For example, in a 50 µl reaction, the total amount of enzyme added should not exceed 5 µl.

l  If two different incubation temperatures are necessary, choose the optimal reaction buffer and set up reaction accordingly. Add the first enzyme and incubate at the desired temperature. Then, heat inactivate the first enzyme, add the second enzyme and incubate at the recommended temperature.

l  Depending on an enzyme's activity rating in a non-optimal EzBuffer, the number of units or incubation time may be adjusted to compensate for the slower rate of cleavage.


[Example] For the performing double digestion reaction using Not I and Pvu II, we recommend to select a EzBuffer II and use double unit of Not I than Pvu II because Not I exhibits only 50% of activity on EzBuffer II. In this case, EzBuffer I, III or IV is not recommended because one of the two enzymes show less than 50% activity in those buffer. Alternatively, FastCut buffer (former name: EZ-One™ buffer) can be used because both enzymes are compatible with FastCut buffer (former name: EZ-One™ buffer) system.

Enzyme

Cat.#

Supplied EzBuffer

% Activity in EzBuffers

I

II

III

IV

Not I

R001

III

0

50

100

0

Pvu II

R021

II

75

100

25

10

 

Setting up a Double Digestion with a Unique EzBuffer

Enzynomics currently supplies 5 enzymes with Unique Ezbuffers: EcoR I, BamH I, Acc III, Bal I, Dpn II. Basically, choose a Unique EzBuffer that results in the most activity for both enzymes by referring to the activity chart of Unique EzBuffer.

 

Setting up a Sequential Digestion

If no common buffer is satisfactory for two-enzyme digestion, DNA digestion should be carried out sequentially. For example, when the two restriction endonucleases have different salt sensitivity, cleave DNA first with a restriction endonuclease that requires a lower salt concentration and then adjust the salt concentration so that the reaction mixture contains optimal salt concentration for the second restriction endonuclease. When star activity of the first enzyme is expected due to a change in salt condition, heat inactivation of the first enzyme is necessary before further digestion with a second restriction enzyme. If the first restriction endonuclease is resistant to heat inactivation, it is recommended to remove the enzyme using phenol/chloroform extraction or a DNA purification kit prior to treating DNA with the second restriction endonuclease.

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