SSR方法

Protocol for SSR

1. Reaction System

：

Constitutes

ddH

2

O

V

olume(ul)

13

Final density

10xPCR buffer(contain 15mM MgCl

2

)

2

1x

dNTP(10mM)

0.4

2mM(each)

Primer F(2uM)

1.5

0.2uM

Primer R(2uM)

1.5

0.2uM

Taq polymerase

0.1

Template

2

50ng-100ng

Total

20

NOTE: Stocking reagent of primer is 100uMol/L, working reagent is 2uMol/L.

2. PCR

Protocol

94

℃，

5mins;

94

℃

,

1

minutes;

55

℃

,40-60

seconds

;

72

℃

,1-2

minutes;

72

℃

,5

minutes;

35 cycles

。

Prepare the plates

3 Wash the glass plates and the comb, then put its at room temperature and air dry.

Note: Clean plates are necessary! Wash the plates with warm soapy water, rinse with

deionized

water

and

then

ethanol,

and

air

dry.

If

the

plates

look

particularly

dirty,

acid-wash them.

4 Assemble the glass plates with spacers for casting the gel. Put the shorter plate in

fovea, the longer plate is the other.

5

Prepare

about

0.8

agarose

gel

(don't

add

the

EB),

inject

it

into

the

gap

that

the

longer plates appear with 1000ul Pipette. Allow it to dry at room temperature for 10

minutes. Then you can inject the prepared acrylamide gel.

Prepare the gel

For example: 80 ml 6% Denaturing Polyacrylamide Gel

6

Urea 12 grams(final concentration is 2.5M. the max can attain to

7M, that is, you can add 33.6 g Urea)

7

8

ml

of

10

x

TBE

electrophoresis

buffer

(final

concentration

is

1

x

TBE

electrophoresis buffer )

8

12 ml of 40% Acrylamide (final concentration is 6% Acrylamide)

9

Add ddH

2

O to total volume 80 ml

1