Cleaving a DNA substrate with two restriction endonucleases simultaneously (double
digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction
conditions that optimize enzyme activity as well as avoid star
activity associated with
some enzymes is an important consideration.
Each enzyme is supplied with its optimal NEBuffer to ensure 100% activity. NEBuffer compositions
are listed on buffer
pages and
on the data card sent with each enzyme. The NEBuffer
Activity Chart rates the
percentage activity of each restriction endonuclease in the four standard NEBuffers.
The NEBuffer that results in the most activity for both enzymes should be used for the
double digest as long as star activity is not a factor. Depending on an enzyme's activity
rating in a non-optimal NEBuffer, the number of units of enzyme or incubation time of
the reaction may be adjusted to compensate for slower rate of cleavage.
If no single NEBuffer can be found to satisfy the buffer requirements of both enzymes,
the reactions can be done sequentially (seq). First, cleave with the restriction endonuclease
that requires the lower salt reaction conditions, then adjust the salt concentration
of the reaction (using a small volume of a concentrated salt solution) to approximate
the reaction conditions of the second restriction endonuclease. Add the second enzyme
and incubate to complete the second reaction. Alternatively, a spin column can be used
to isolate the DNA prior to the second reaction.
Performing double digests with enzymes that are supplied with their own unique NEBuffer and not
one of the four standard NEBuffers is also simple. In most cases, double digests using any of these
enzymes with an enzyme that is supplied with one of the four standard NEBuffers can be done in the
unique NEBuffer. This will ensure that the enzyme with the more specific buffer requirements will
work optimally. Please check the activity ratings in unique
buffers for the most commonly used enzymes.
When using restriction endonucleases in non-optimal NEBuffers, more enzyme or longer digestion time
may be needed to compensate for the slower rate of cleavage under those conditions. Check the NEBuffer
Activity Chart to see how well the second enzyme performs in the salt range of the unique NEBuffer.
Please click here to request the 2007 NEBuffer Activity Chart Poster
Please use NEB Double Digest Finder to
perform a double digest calculation. |
Suggested NEBuffers for Double Digestion
 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | Enzyme | | AatII | AvrII | BamHI | BglII | BsgI | EagI | EcoRI | EcoRV | HindIII | KpnI | MseI | NcoI | NdeI | NheI | NotI | PstI | PvuI | SacI | SacII | SalI | SmaI | SpeI | SphI | XbaI | XhoI | XmaI | | NEBuffer | 4 | 2 | 3 | 3 | 4 | 3 | EcoRI | 3 | 2 | 1 | 2 | 3 | 4 | 2 | 3 | 3 | 3 | 1 | 4 | 3 | 4 | 2 | 2 | 2 | 2 | 4 | | AatII | 4 | -- | 4 | seq | seq | 4 | seq | seq | 4 | 4 | 4 | 4 | 4 | 4 | 4 | seq | 4 | seq | 4 | 4 | seq | 4 | 4 | 4 | 4 | 4 | 4 | | AvrII | 2 | 4 | -- | 3 | 2 | 4 | 3 | EcoRI | 2 | 2 | 1 | 2 | 2 | 4 | 2 | 3 | 3 | 2 | 1 | 4 | 3 | 4 | 2 | 2 | 2 | 2 | 4 | | BamHI | 3 | seq | 3 | -- | 3 | 3 | 3 | EcoRI | 3 | seq | seq | 3 | 3 | 3 | seq | 3 | 3 | 3 | seq | seq | 3 | seq | seq | 3 | 3 | 3 | seq | | BglII | 3 | seq | 2 | 3 | -- | 3 | 3 | EcoRI | 3 | 2 | 2 | 2 | 3 | 3 | 2 | 3 | 3 | 3 | 2 | seq | 3 | seq | 2 | 2 | 2 | 3 | seq | | BsgI | 4 | 4 | 4 | 3 | 3 | -- | seq | seq | 4 | 2 | seq | 2 | 4 | 4 | 4 | 3 | 3 | 3 | 4 | 4 | 3 | 4 | 2 | 4 | 2 | 4 | 4 | | EagI | 3 | seq | 3 | 3 | 3 | seq | -- | EcoRI | 3 | seq | seq | 3 | 3 | 3 | seq | 3 | 3 | 3 | seq | seq | 3 | seq | seq | 3 | 3 | 3 | seq | | EcoRI | EcoRI | seq | EcoRI | EcoRI | EcoRI | seq | EcoRI | -- | EcoRI | EcoRI | 1 | EcoRI | EcoRI | EcoRI | EcoRI | EcoRI | EcoRI | EcoRI | 1 | EcoRI | EcoRI | seq | EcoRI | EcoRI | seq | EcoRI | seq | | EcoRV | 3 | 4 | 2 | 3 | 3 | 4 | 3 | EcoRI | -- | 2 | 2 | 2 | 3 | 2 | 2 | 3 | 3 | 3 | 2 | 2 | 3 | 4 | 2 | 2 | 2 | 3 | 4 | | HindIII | 2 | 4 | 2 | seq | 2 | 2 | seq | EcoRI | 2 | -- | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | seq | 4 | 2 | 2 | 2 | 2 | seq | | KpnI | 1 | 4 | 1 | seq | 2 | seq | seq | 1 | 2 | 2 | -- | 1 | 1 | 1 | 1 | 2 | 1 | 2 | 1 | 4 | seq | seq | 1 | 1 | 2 | 1 | 4 | | MseI | 2 | 4 | 2 | 3 | 2 | 2 | 3 | EcoRI | 2 | 2 | 1 | -- | 2 | 2 | 2 | 2 | 3 | 3 | 2 | 2 | 3 | 4 | 2 | 2 | 2 | 2 | 4 | | NcoI | 3 | 4 | 2 | 3 | 3 | 4 | 3 | EcoRI | 3 | 2 | 1 | 2 | -- | 4 | 2 | 3 | 3 | 3 | 1 | 4 | 3 | 4 | 2 | 2 | 2 | 2 | 4 | | NdeI | 4 | 4 | 4 | 3 | 3 | 4 | 3 | EcoRI | 2 | 2 | 1 | 2 | 4 | -- | 4 | 3 | 3 | 3 | 4 | 4 | 3 | 4 | 2 | 2 | 2 | 4 | 4 | | NheI | 2 | 4 | 2 | seq | 2 | 4 | seq | EcoRI | 2 | 2 | 1 | 2 | 2 | 4 | -- | 2 | 2 | 2 | 1 | 4 | seq | 4 | 2 | 2 | 2 | 2 | 4 | | NotI | 3 | seq | 3 | 3 | 3 | 3 | 3 | EcoRI | 3 | 2 | 2 | 2 | 3 | 3 | 2 | -- | 3 | 3 | 2 | 2 | 3 | seq | 2 | 2 | 3 | 3 | 2 | | PstI | 3 | 4 | 3 | 3 | 3 | 3 | 3 | EcoRI | 3 | 2 | 1 | 3 | 3 | 3 | 2 | 3 | -- | 3 | 1 | 2 | 3 | 4 | 2 | 2 | 3 | 3 | 4 | | PvuI | 3 | seq | 2 | 3 | 3 | 3 | 3 | EcoRI | 3 | 2 | 2 | 3 | 3 | 3 | 2 | 3 | 3 | -- | 2 | 2 | 3 | seq | 2 | 2 | 3 | 3 | seq | | SacI | 1 | 4 | 1 | seq | 2 | 4 | seq | 1 | 2 | 2 | 1 | 2 | 1 | 4 | 1 | 2 | 1 | 2 | -- | 4 | seq | 4 | 1 | 1 | 4 | 1 | 4 | | SacII | 4 | 4 | 4 | seq | seq | 4 | seq | EcoRI | 2 | 2 | 4 | 2 | 4 | 4 | 4 | 2 | 2 | 2 | 4 | -- | seq | 4 | 2 | 4 | 2 | 4 | 4 | | SalI | 3 | seq | 3 | 3 | 3 | 3 | 3 | EcoRI | 3 | seq | seq | 3 | 3 | 3 | seq | 3 | 3 | 3 | seq | seq | -- | seq | seq | 3 | 3 | 3 | seq | | SmaI | 4 | 4 | 4 | seq | seq | 4 | seq | seq | 4 | 4 | seq | 4 | 4 | 4 | 4 | seq | 4 | seq | 4 | 4 | seq | -- | 4 | 4 | 4 | 4 | 4 | | SpeI | 2 | 4 | 2 | seq | 2 | 2 | seq | EcoRI | 2 | 2 | 1 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 1 | 2 | seq | 4 | -- | 2 | 2 | 2 | 4 | | SphI | 2 | 4 | 2 | 3 | 2 | 4 | 3 | EcoRI | 2 | 2 | 1 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 1 | 4 | 3 | 4 | 2 | -- | 2 | 2 | 4 | | XbaI | 2 | 4 | 2 | 3 | 2 | 2 | 3 | seq | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 3 | 3 | 3 | 4 | 2 | 3 | 4 | 2 | 2 | -- | 2 | 4 | | XhoI | 2 | 4 | 2 | 3 | 3 | 4 | 3 | EcoRI | 3 | 2 | 1 | 2 | 2 | 4 | 2 | 3 | 3 | 3 | 1 | 4 | 3 | 4 | 2 | 2 | 2 | -- | 4 | | XmaI | 4 | 4 | 4 | seq | seq | 4 | seq | seq | 4 | seq | 4 | 4 | 4 | 4 | 4 | 2 | 4 | seq | 4 | 4 | seq | 4 | 4 | 4 | 4 | 4 | -- |
Double Digest Notes:
- If BSA is a buffer requirement for either enzyme, add it to the double digest reaction. BSA will not inhibit
any restriction enzyme.
- The final concentration of glycerol in any reaction should be less than 5% to minimize the possibility
of star activity. To avoid this situation, an increase in total reaction volume may be necessary.
- Double digestion is not recommended for certain enzyme combinations. In these cases a sequential digest
is required. In the table above, these combinations are indicated by the abbreviation "seq".
- This data is based on 1-2 hour digests. Overnight double digests should be avoided due to possible star
activity.
- BsgI requires SAM. SAM as an additive does not have a negative affect on activity
of other enzymes.
- SmaI is incubated at 25°C; then raise the temperature to 37°C to allow your second enzyme to work.
SmaI may be heat killed between the digests.
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Double Digest FAQ Spotlight:
Q: I would like to digest DNA with EcoRI and XbaI at the same time. The Double Digest Finder recommends a
sequential digest, using each enzyme in its supplied NEBuffer. However, both of these enzymes show 100% activity
in NEBuffer 2. Why is a double digest not recommended with these enzymes?
A: Although EcoRI shows 100% activity in NEBuffer 2, it also exhibits significant star activity in this buffer,
resulting in non-specific, unintended cleavage. This is also observed when using NEBuffer 4. For this reason,
a sequential digest is recommended. However, research has shown that EcoRI exhibits less star activity in
NEBuffers 1 and 3. Since XbaI has 75% activity in NEBuffer 3 a double digest could be performed in this buffer,
provided that the reactions are set up according to the recommended reaction conditions for avoiding star
activity. |
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