superiority基因敲除详细步骤

The

following

protocols

take

MLCK

(myosin

light

chain

kinase)

as

an

example.

General steps:

1.

BAC extraction (It is necessary for us to identify the BAC by PCR)

2.

Transform BAC to EL350 ( Cm+)

3.

Retrieving (Cm+

Amp+)

4.

Targeting 1st

lox

P (Amp+

Amp+ and K+)

5.

Transform MLCK 1st

lox

P to EL350 to get purify MLCK 1st

lox

P ( Amp+ and K+)

6.

MLCK 1st

lox

P pop out (Amp+ and K+

AmP+)

7.

Transform MLCK 1st

lox

P pop out to EL250 (Amp+)

8.

Targeting 2nd

lox

P (Amp+

Amp+ and K+)

9.

Transform MLCK 2nd

lox

P to DH-5

α

or XL

1

-Blue ( Amp+ and K+)

10.

Linearization

1. BAC extraction

Solution I:

0.025 M

EDTA

0.01M

Glucose

0.05M

pH 8.0

Solution II:

SDS

NaOH

1 %

0.2M

fresh prepared (1V

olume 2% SDS + 1V

olume 0.4M NaOH)

Solution III:

(120 ml

5 M KAc

+

23 ml HAc

+

57 ml H2O) / 200 ml

Protocol:

1

1.

Harvest 50 ml bacterial cells (O/N) by centrifugation at 9,000 r/m for 10 min, pour off the

supernatant clearly.

2.

Add 5ml ice- cold Solution I, mix.

3.

Add 10 ml Solution II, invert several times gently.

4.

Add 7.5 ml Solution III, invert several times gently.

5.

Centrifuge at 9,000 r/min for 10 min at 4

℃

. Remove the supernatant to a fresh tube.

6.

Add 0.6 volume of isopropanol

7.

Centrifuge at 9,000 r/min for 10 min at 4

℃

. Remove supernatant.

8.

Dissolve DNA pellet in 400 ul TE, add 20 ul 10mg/ml RNase, 55

℃

, 20-30min.

9.

Add equal volume of Phenol/chloroform (1:1), mix and centrifuge at 12,500 rpm for 5 min at

RT. (From this step, 1.5 ml tube can be used)

10.

Transfer supernatant to a fresh tube, add equal volume of chloroform, mix and centrifuge at

12,500 rpm for 10min at RT

11.

Add 0.1 volume of 3M NaAc

(pH5.3) and 2 volume of ethanol (stored at

-20

℃

). Mix and

centrifuge at 12,500 rpm for 10 min at 4

℃

.

12.

Dissolve DNA with 50 ul pH 7.4 MilliQ H2O.

(

TENS

isn’t good for BAC extraction)

2. Transform BAC into EL350

(Cm+)

1.

Pick up a single colony of EL350 to 3ml LB, grow at 32

℃

O/N (12-16h)

2.

Next day, incubate 1 ml of O/N culture to 50 ml LB,

32

℃

for 2-3h to

OD600=0.5

From this step, all on ice or in 4

℃

3.

Transfer 6ml cells to 15 ml centrifuge tube, put on ice for

15 min

4.

Spin at 3,500 rpm for 6 min, resuspend cell with 1.5ml pH 7.4 MilliQ H2O.

5.

Spin, wash twice more.

6.

Remove supernatant, add about 50 ul pH 7.4 MilliQ H2O.

7.

Add 10 ul BAC to 50 ul competent cells, pipette them to an electroporation cup (0.1 cM gap).

8.

1.75kV

, 25 uF, 200 ohms.

9.

Add 1ml SOC to each curvette

and incubate at 32

℃

for 1h.

10. Centrifuge at 3,500 rpm for 3 min, pour off most of the supernant and plate cells (Cm+).

2

3. Retrieving (Cm+

Amp+)

Retrieval plasmid construction

1.

PCR amplify two 500 bp homologous arms

（

BAC as template

）

2.

Insert these two fragments to T vector

3.

Not

I ,

Hind

III cutting and

Spe

I

Hind

III cutting 500 bp fragments ligated with

Not

I ,

Spe

I

cutting

PL253

4.

Transform, identify the positive colones.

5.

Cut the retrieval plasmid with

Hind

III, purify the product.

Transformation

1.

Pick up a single colony of BAC-EL350 to 3ml LB, grow at 32

℃

O/N (12-16h)

2.

Next day, incubate 1 ml of O/N culture to 50 ml LB, 32

℃

for 2-3h to OD600=0.5

3.

Induce BAC- EL350 in 42

℃

water bath by shaking for 15 min.

From this step, all on ice or in 4

℃

4.

Transfer 6ml cells to 15 ml centrifuge tube, put on ice for 15 min

5.

Spin at 3,500 rpm for 6 min, resuspend cells with 1.5ml pH 7.4 MilliQ

6.

Spin, wash twice more.

7.

Remove supernatant, add about 50

μl

pH 7.4 MilliQ

8.

Add 200-500ng linear retrieval plasmid to 50ul BAC-EL350, pipette them to an electroporation

cup (0.1 cM gap).

9.

1.75kV

, 25 uF, 200 ohms.

10.

Add 1ml SOC to each curvette and incubate at 32

℃

for 1h.

11.

Centrifuge at 3,500 rpm for 3 min, pour off most of the supernatant and plate cells (Amp+).

3

Retrieving

Cm

+

24

25

26

27

28

29

C< /p>

m

MLCK-BAC

EL350, 42

℃

PL253 retrieving vector< /p>

A

m

p

+

Amp

+

24

25< /p>

26

27

28

2 9

C

m

MLCK- PL253

MLCK-BAC

MLCK-PL253 digestion map

4. Targeting 1st

lox

P(Amp+

Amp+ and K+)

Mini-targeting vector construction

1.

PCR amplify 80 bp homologous arms

lox

P floxed

Neo

cassette (1st

lox

P) from PL452.

2.

Ligate

1st

lox

P

with

T

vector

(If

the

PCR

product

quantity

is

enough

for

later

use

,

this

procedure is not necessary)

3.

Cut 1st

lox

P

–

T to get 1

st

lox

P fragment, purify the product.

Note: If you use the ligation method to construct the mini-targeting vector, two ~500bp homologous

arms

are

ligated

to

the

floxed

Neo

cassette

excised

from

PL452

with

Eco

RI

and

Bam

HI

and

to

PL253 that is linearized by

Not

I and

Sal

I

.

For more detail, pls see, Lee, E. C.

et al

. A highly efficient Escherichia coli-based chromosome

engineering

system adapted for recombinogenic targeting and subcloning of BAC DNA.

Genomics

73

,

56-65 (2001)

Transformation

1.

Pick up a single colony of MLCK- PL253-EL350 to 3ml LB, grow at 32

℃

O/N (12-16h)

2.

Next day, incubate 1 ml of O/N culture to 50 ml LB, 32

℃

for 2-3h to OD600=0.5

3.

Induce MLCK- PL253-EL350 in 42

℃

water bath by shaking for 15 min.

From this step, all on ice or in 4

℃

4

4.

Transfer 6ml cells to 15 ml centrifuge tube, put on ice for 15 min

5.

Spin at 3500 rpm 6 min, resuspend cell with 1.5ml pH 7.4MilliQ

6.

Spin ,wash twice more.

7.

Remove supernatant, add about 50

μl

pH 7.4MilliQ

8.

Add 200ng 1st

lox

P fragment to 50ul MLCK- PL253-EL350, pipette them to an electroporation

cup (0.1 cM gap).

9.

1.75kV

, 25 uF, 200 ohms.

10.

Add 1ml SOC to each curvette and incubate at 32

℃

for 1h.

11.

Centrifuge at 3,500 rpm for 3 min, pour off most of the supernatant and plate cells (Amp+ and

K+).

Targeting 1stLoxP

25

< br>26

27

Amp+

A

p

+

MLCK- PL253

EL350, 42

℃

Lo xp

floxed

Neo cassette

Neo

Amp+ K+

A

m

p

+

M LCK PL253

digestion map

MLCK-PL253 1stLoxP

MLCK- PL253 1stLoxP

digestion map

H indIII

EcoR

I

EcoR

I

5.

Transform MLCK 1st

lox

P to EL350 to get purified MLCK 1st

lox

P ( Amp+

and K+)

Note:

EL350

contains

multiple

copies

of

plasmids,

so

there

will

be

both

recombinant

and

unrecombinant plasmids in EL350.

6. 1st

lox

P pop out (Amp+ and K+

Amp+)

5