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DNA Restriction digest

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Introduction:

DNA restriction endonucleases are mainly used for cutting DNA to yield sticky ends for directional ligation into cloning vectors. These enzymes are actually classed as type II  restriction endonucleases and they cut at specific sites internal to the DNA strand. For example one commonly used enzyme BamHI recognises the site GGATCC and cleaves duplex with this sequence to give a sticky end with a 5' overhang:

BamHI-cutsite

As with most enzymes, the restriction endonucleases have varying activity depending on the buffer they reside in and with this in mind it is definitely worth consulting the following tables which describe buffer systems from each supplier (Promega, NEB etc.) and compatible buffer systems for double digests (i.e. using two different enzymes in the same reaction).

Promega

Buffer

pH
(at 37°C) 

Tris-HCl
(mM)

MgCl2
(mM)

NaCl
(mM)

KCl
(mM)

DTT
(mM)

  A

7.5

6

6

6

-

1

  B 

7.5

6

6

50

-

1

  C

7.9

10

10

50

-

1

  D

7.9

6

6

150

-

1

  E

7.5

6

6

100

-

1

  F

8.5

10

10

100

-

1

  G

8.2

50

5

-

-

-

  H

7.5

90

10

50

-

-

  J

7.5

10

7

-

50

1

  K

7.4

10

10

-

150

-

  L

9.0

10

3

100

-

-

NEB

NEBuffer 1 (yellow):

10 mM Bis Tris Propane-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.0 at 25°C).

NEBuffer 2 (blue):

10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT (pH 7.9 at 25°C).

NEBuffer 3 (red):

50 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT (pH 7.9 at 25°C).

NEBuffer 4 (green):

20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT (pH 7.9 at 25°C).

Unique Buffers:

BamHI – 150mM NaCl, 10mM Tris, pH 7.9, 10mM MgCl2, 1mM DTT.
EcoRI – 50mM NaCl, 100mM Tris, pH 7.5, 10mM MgCl2, 0.025% Triton X100
SalI – 150mM NaCl, 10mM Tris, pH 7.9, 10mM MgCl2, 1mM DTT

NEB Buffer and Enzyme Compatability chart:

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 4 3 3 4 3 EcoRI 3 2 1 4 3 4 2 3 3 3 1 4 3 4 4 2 4 4 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 4 4 -- 3 2 4 3 EcoRI 2 2 1 4 4 4 2 3 3 2 1 4 3 4 4 2 4 4 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 2 3 seq 2 2 2 3 2
BsgI 4 4 4 3 3 -- seq seq 4 2 seq 4 4 4 4 3 3 3 4 4 3 4 4 4 4 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 seq 1 EcoRI EcoRI EcoRI 1 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 seq 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 4 4 4 3 2 4 3 EcoRI 2 2 1 -- 4 4 2 2 3 3 4 4 3 4 4 2 4 4 4
NcoI 3 4 4 3 3 4 3 EcoRI 3 2 1 4 -- 4 2 3 3 3 1 4 3 4 4 2 4 4 4
NdeI 4 4 4 3 3 4 3 EcoRI 2 2 1 4 4 -- 4 3 3 3 4 4 3 4 4 2 4 4 4
NheI 2 4 2 seq 2 4 seq 1 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 2
SacI 1 4 1 seq 2 4 seq 1 2 2 1 4 1 4 1 2 1 2 -- 4 seq 4 1 1 4 1 4
SacII 4 4 4 seq 2 4 seq EcoRI 2 2 4 4 4 4 4 2 2 2 4 -- seq 4 4 4 4 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 4 4 4 seq 2 4 seq EcoRI 2 2 1 4 4 4 2 2 2 2 1 4 seq 4 -- 2 4 4 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 4 4 4 3 2 4 3 seq 2 2 2 4 4 4 2 3 3 3 4 4 3 4 4 2 -- 4 4
XhoI 4 4 4 3 3 4 3 EcoRI 3 2 1 4 4 4 2 3 3 3 1 4 3 4 4 2 4 -- 4
XmaI 4 4 4 seq 2 4 seq seq 4 seq 4 4 4 4 4 2 4 2 4 4 seq 4 4 4 4 4 --


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