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Double Digests

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 AatIIAvrIIBamHIBglIIBsgIEagIEcoRIEcoRVHindIIIKpnIMseINcoINdeINheINotIPstIPvuISacISacIISalISmaISpeISphIXbaIXhoIXmaI
NEBuffer423343EcoRI3212342333143422224
AatII4--4seqseq4seqseq4444444seq4seq44seq444444
AvrII24--3243EcoRI2212242332143422224
BamHI3seq3--333EcoRI3seqseq333seq333seqseq3seqseq333seq
BglII3seq23--33EcoRI32223323332seq3seq2223seq
BsgI44433--seqseq42seq2444333443424244
EagI3seq333seq--EcoRI3seqseq333seq333seqseq3seqseq333seq
EcoRIEcoRIseqEcoRIEcoRIEcoRIseqEcoRI--EcoRIEcoRI1EcoRIEcoRIEcoRIEcoRIEcoRIEcoRIEcoRI1EcoRIEcoRIseqEcoRIEcoRIseqEcoRIseq
EcoRV3423343EcoRI--222322333223422234
HindIII242seq22seqEcoRI2--2222222222seq42222seq
KpnI141seq2seqseq122--111121214seqseq11214
MseI2423223EcoRI221--222233223422224
NcoI3423343EcoRI3212--42333143422224
NdeI4443343EcoRI22124--4333443422244
NheI242seq24seqEcoRI221224--22214seq422224
NotI3seq33333EcoRI3222332--33223seq22332
PstI3433333EcoRI32133323--3123422334
PvuI3seq23333EcoRI322333233--223seq2233seq
SacI141seq24seq12212141212--4seq411414
SacII444seqseq4seqEcoRI22424442224--seq424244
SalI3seq33333EcoRI3seqseq333seq333seqseq--seqseq333seq
SmaI444seqseq4seqseq44seq4444seq4seq44seq--44444
SpeI242seq22seqEcoRI221222222212seq4--2224
SphI2423243EcoRI221222222214342--224
XbaI2423223seq2222222333423422--24
XhoI2423343EcoRI32122423331434222--4
XmaI444seqseq4seqseq4seq4444424seq44seq44444--


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.
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.