實(shí)驗(yàn)生化圖片yu plasmid

BamH

ICD

(1.2

kb)TK

(1.3

kb)Purification

and

Identification

of

PlasmidDNAStructure

of

pEC-CT8

kbIntroductionpEC-ctplasmid:8

kb

in

lengthcarry

genes

CD

(1.2kb)

and

TK(1.3kb)single

endonuclease

sites

for

EcoR

I,

BamHI,Hind

IIIBamH

IEcoR

IHind

IIIIntroduction8

kbEcoR

IBamH

IHind

IIIHind

III

8kbHind

III

and

BamH

I1.2

kb

6.8

kbC

DProcedure:1.

Isolation

and

purification

of

plasmid

DNA(1)

Culture

of

bacteria:0.3

ml

of

the

stored

bacteriasample↓is

inoculated

into

the

LB

liquid

culture

medium

(ampicillin

50

mg/ml)↓incubated

at

37

C

overnight

(about

14-18

h)↓Take

3

ml

of

the

above

culture

to

inoculate

into

60

ml

of

LB↓Shake

at

200

rpm,37

C

for

4-6

h

(

A580

=0.4-0.6)↓Add

chloramphenicol

to

the

culture

medium(final

con.

of

40

mg/ml)↓continue

the

incubation

for

15-17

h↓Take

6

clean

eppendorf

tubes

of

1.5

ml↓transfer

1.4

ml

of

the

above

bacteria

culture

to

each

tube↓centrifuge

at

9000rpm

for

1min↓

discard

the

supernatantprecipitateAdd

0.8

ml

of

STE

solution

to

a

tube

to

wash

the

bacteria↓then

transfer

all

to

the

second

tube↓and

do

so

one

by

one

until

the

last

tube↓centrifuge

at

12000

rpm

for

30

secs.↓precipitate(2)

Isolation

and

purification

of

plasmid

DNARe-suspend

the

precipitate

in

200

ul

of

solution

I↓mix

with

tipfor

1

min↓Add

400

ul

of

0.2

N

NaOH-1%

SDS

solution‖↓*

mix

byinverting

thetube5-10times↓stand

for 5

min.

at

room

temperatureNote:

mixing

with

strong

vibration

should

be

avoided↓Add

300

ul

of

solutionIII↓*mixwell

by

inverting

the

tube

5-10

times,

stand

for

10

min.atRTRemove

proteinsCentrifuge

at

12000

rpm

for

3

min.↓Carefully

transfer

the

supernatant

into

another

tube↓add

0.6

volume

of

isopropanol

(about

540

ul)↓mix

by

inverting

the

tube

several

times↓at

room

temperature

for

10

min.↓Centrifuge

at

12000

rpm,

for

5

min↓discard

the

supernatantPrecipitate

plasmidsThe

precipitate

is

dried

at

room

temperature

(for

about

10-15min)↓Add

0.5

ml

of

TE

buffer

solution

to

dissolve

the

precipitate↓Add

equal

volume

of

saturated

phenol

solution↓vortex

for

1

min↓centrifuge

at

12000rpm

for

3

min↓transfer

the

supernatant

into

another

tube↓extract

it

with

chloroform-isoamyl(equal

volume

)↓mix

thoroughly

for

1min↓centrifuge

at

12000rpm

for

3

minPrecipitate

proteinsCollect

the

upper

aqueous

layer

to

another

clean

tube↓add

2.5*

volumes

of

pre-cooled

anhydrous

ethanol↓Let

it

stay

at

-20 C

for

30

min

(or

more)↓centrifuge

at

12000rpm

for

10

min.discard

the

supernatant

↓dry

the

precipitate

at

room

temperature↓Add

100

ul

of

TE

buffer

solution

to

thoroughly

dissolvethe

precipitatePrecipitate

plasmids2.

Determination

of

DNA

concentration

in

samplespectrophotometerethidium

bromide

(EB)

semi-quantitative

measurementProcedure:agarose

gel

preparation

(

TBE

buffer)make

a

series

of

standard

DNA

solutions standard

DNA

solutions

:1.00,

0.50,

0.25,

0.10,

0.05,

0.025

and

0.01

mg/mlapply

1

ul

of

each

concentration

of

the

standards

onto

theagarose

gel.↓apply

1

ul

of

the

sample

DNA

solution

onto

the

agarose

gel.↓1

hr

after

sample

application↓the

fluorescence

spots

can

be

observed

under

uv

light(the

sample

DNA

concentration

is

estimated

by

comparing

its

fluorescentdensity

with

those

of

the

standards)standard

DNA

solutionsyour

sample0.01

0.025

0.05 0.10

0.25

0.50

1.003.

Endonuclease

digestion

of

plasmid

DNA(1)

Generation

of

linear

plasmid

DNA

(

0.5ml

eppendorf

tube):mix,

incubate

at

37 C

for

2

hSample

5ul10x

buffer 2.5

ulHind

III

1ulDDH2O 15

ul(2)

Generation

of

apoB

gene

PEC-CT

fragment

(

0.5ml

eppendorftube):mix,

incubate

at

37 C

for

2

hSample10x

bufferHind

IIIBamH

IDDH2O5ul2.5

ul1ul1

ul15ul4.

Identification

of

plasmid

DNA0.8%

agarose

gel

preparation

(

TBE

buffer)Sample

application:take

10

ul

of

each

sample,

mix

with

2

ul

of

loading

bufferThe

samples

to

be

applied

should

include:plasmid

extractHind

III

digest

of

the

plasmidHindIII

+

BamH

I

digest

of

the

plasmidMarker

(DL15000):15000,10000,7500,5000,2500,1000,2500.8%

agarose

gel--+1

2

3

4(3)

Electrophoresis:120V

for

5

min

until

the

samples

enter

into

the

gel,andthenchangethevoltage

to

100

V

for

about25~30min.(4)

Identification

of

DNA

bands:Observe

the

DNA

bands

and

take

the

photographs

directly

on

a

UVtransilluminator

that

is

connectedto

animage

analysis