pcr保护碱基

Cleavage Close to the End of DNA Fragments

(linearized vector)

Linearized

vectors

were

incubated

with

the

indicated

enzymes

(10

units/?

g)

for

60

minutes

at

the

recommended

incubation

temperature

and

NEBuffer

for

each

enzyme.

Following ligation and transformation, cleavage efficiencies were determined by dividing

the

number

of

transformants

from

the

digestion

reaction

by

the

number

obtained

f

rom

religation of the linearized DNA (typically 100-500 colonies) and subtracting from 100%.

recognition

site

and

the

terminus

of

the

fragment;

this

number

does

not

include

the

single-stranded overhang from the initial cut. Since it has not been demonstrated whether

these

single- stranded

nucleotides

contribute

to

cleavage efficiency,

4 bases

should

be

added to the indicated numbers when designing PCR primers. Average efficiencies were

rounded

to

the

nearest

whole

number;

experimental

variation

was

typically

within

10%.

The numbers in parentheses refer to the number of independent trials for each enzyme

tested (from Moreira, R. and Noren, C. (1995), Biotechniques, 19, 56-59).

Note: As a general rule, enzymes not listed below require 6 bases pairs on

either side of their recognition site to cleave efficiently.

|

A

|

B

|

E

|

H

|

K

|

M

|

N

|

P

|

S

|

X

|

Base pairs

%Cleavage

Enzyme

from End

Aat II

3

2

1

Efficiency

88 (2)

100 (2)

95 (2)

99 (2)

75 (3)

13 (2)

100 (1)

100 (2)

100 (1)

97 (2)

100 (2)

97 (2)

100 (2)

100 (2)

100 (1)

Vector

LITMUS 29

LITMUS 28

LITMUS 29

LITMUS 29

pNEB193

LITMUS 29

LITMUS 29

LITMUS 29

LITMUS 38

pNEB193

LITMUS 29

LITMUS 29

LITMUS 29

LITMUS 29

LITMUS 39

Initial Cut

Nco I

Nco I

PinA I

Spe I

Sac I

Stu I

Xba I

Aat II

Spe I

BamH I

Sac I

Hind III

Nsi I

BssH II

BsrG I

Acc65 I

2

1

Afl II

Age I

1

1

1

Apa I

Asc I

Avr II

BamH I

Bgl II

BsiW I

BspE I

2

1

1

1

3

2

2

1

8 (2)

99 (2)

88 (2)

100 (2)

100 (2)

100 (1)

LITMUS 38

LITMUS 39

LITMUS 38

LITMUS 29

LITMUS 39

LITMUS 29

LITMUS 29

LITMUS 39

LITMUS 29

LITMUS 29

LITMUS 28

LITMUS 29

LITMUS 38

LITMUS 38

LITMUS 29

LITMUS 29

pNEB193

LITMUS 39

LITMUS 39

LITMUS 28

LITMUS 39

LITMUS 39

LITMUS 39

Bluescript SK-

Bluescript SK-

Bluescript SK-

LITMUS 29

LITMUS 29

LITMUS 28

pNEB193

pNEB193

LITMUS 29

LITMUS 39

BsrG I

Sph I

BspE I

BsiW I

Nhe I

Xho I

Pst I

Nhe I

Pst I

Nco I

Nco I

BamH I

NgoM IV

Hind III

Spe I

Sac I

Sac I

Eag I

NgoM IV

Hind III

Mun I

EcoR I

Eag I

Spe I

Ksp I

Xba I

BssH II

Bgl II

BssH II

BamH I

Pst I

EcoR V

Hind III

BsrG I

2

1

BssH II

Eag I

EcoR I

2

2

1

1

1

88 (1)

100 (1)

100 (2)

90 (2)

91 (2)

0 (2)

97 (1)

93 (1)

100 (2)

100 (2)

99 (2)

99 (2)

100 (1)

100 (1)

100 (1)

100 (1)

82 (1)

100 (2)

100 (1)

EcoR V

Hind III

1

3

2

1

Kas I

2

1

Kpn I

2

2

1

Mlu I

Mun I

Nco I

NgoM IV

Nhe I

2

2

2

2

1

2

Not I

7

4

1

98 (2)

100 (2)

77 (4)

95 (2)

76 (3)

94 (2)

98 (1)

50 (5)

Nsi I

3

3

2

Pac I

Pme I

Pst I

1

1

3

2

1

37 (3)

99 (2)

89 (2)

23 (2)

61 (3)

100 (2)

100 (2)

99 (1)

97 (1)

92 (2)

99 (2)

94 (1)

97 (2)

98 (1)

92 (1)

LITMUS 29

LITMUS 29

LITMUS 39

LITMUS 39

LITMUS 38

LITMUS 29

LITMUS 29

LITMUS 39

LITMUS 39

LITMUS 38

LITMUS 29

LITMUS 29

LITMUS 29

pNEB193

pNEB193

EcoR I

Avr II

Spe I

Sph I

Sph I

Acc65 I

Kpn I

Sal I

BsrG I

Sal I

Age I

PinA I

EcoR I

Asc I

BssH II

Sac I

Sal I

1

3

2

1

Spe I

2

2

Sph I

2

2

1

Xba I

1

1

Xho I

Xma I

1

2

2

New England Biolabs T

echnical Liter

ature - Updated

03/05/2004

Cleavage Close to the End of DNA Fragments

(oligonucleotides)

To test the varying requirements restriction endonucleases have for the number of bases

flanking their recognition sequences, a series of short, double-stranded oligonucleotides

that contain the restriction endonuclease recognition sites (shown in red) were digested.

This information may be helpful when choosing the order of addition of two restriction

endonucleases for a double digest (a particular concern when cleaving sites close

together in a polylinker), or when selecting enzymes most likely to cleave at the end of a

DNA fragment.

The experiment was performed as follows: 0.1 A

260

unit of oligonucleotide was

phosphorylated using T4 polynucleotide kinase and

?

- [

32

P] ATP. 1 ?

g of 5?

[

32

P]-labeled

oligonucleotide was incubated at 20°

C with 20 units of restriction endonuclease in a buffer

containing 70 mM Tris-HCl (pH 7.6), 10 mM MgCl

2

, 5 mM DTT and NaCl or KCl

depending on the salt requirement of each particular restriction endonuclease. Aliquots

were taken at 2 hours and 20 hours and analyzed by 20% PAGE (7 M urea). Percent

cleavage was determined by visual estimate of autoradiographs.

As a control, self-ligated oligonucleotides were cleaved efficiently. Decreased cleavage

efficiency for some of the longer palindromic oligonucleotides may be caused by the

formation of hairpin loops

.