SSR技术原理,方法及步骤

模板

DNA 1ul (30-50ng)

ISSR

引物

1ul (

约

5pmol)

10xPCR Buffer 2.5ul

MgCl

2

2ul

dNTP 2ul

Taq

酶

1

单位（

U

）

加

ddH

2

O

至

25ul

混匀稍离心

,

加一滴（约

20 ul

）矿物油。

2.

在

PCR

仪中预变性

94

℃

2

分钟

,

然后循环

: 94

℃

1

分钟，

45

℃

-68

℃

40

秒，

72

℃

1-2

分钟，共

40

轮循环。

3.

循环结束后

, 72

℃

10

分钟，

< br>4

℃保存。

4.

取

PCR

产物

15ul

< br>加

3ul

上样缓冲液

(6x)< /p>

于

1.6%

或

1.8%

琼脂糖胶上电泳

,

稳压

50-100

Ｖ（电压低带型整齐，

分辨率高）。

5.

电 泳结束，溴化乙锭染色

20

分钟。

6.

用凝胶成像仪观察、拍照。

操作流程简图：

3. SSR GEL and Silver Staining Protocol

Paula Marquardt & Craig Echt

Published in: Echt CS, May- Marquardt P, Hseih M, Zahorchak R. 1996. Characterization of

microsatellite markers in eastern white pine.

Genome 39:1102-1108

.

Comments can be directed to Paula Marquardt at:

USDA Forest Service Research

5985 Highway K

;.

.

Rhinelander, WI 54501

USA

Phone: 715-362-1121

Fax: 715-362-1166

e-mail: pmarquar@

I. EQUIPMENT:

DNA sequencing unit (35 x 45 cm) & 2000V power supply

Clamps

Lg. plastic trays (4), about 43 x 50 x 8 cm, and one lid

Two rocking platforms

Heat block for microtiter plates. A microplate vortexer is helpful.

II.

MOLD ASSEMBLY:

Notes:

Bind silane is toxic and should be used in a chemical fume hood. Wear gloves when

handling this solution. Use a small piece of vinyl tape on a lower outside corner of the acrylease

treated glass gel plate to mark the untreated side and also help distinguish the plates. This helps

avoid confusion between plates when using offset plates. The tape can remain in place through

several electrophoresis / washing cycles.

1.

Wash

inner

and

outer

plates

well

with

alconox

cleanser.

Rinse

well

with

tap

water,

deionized or distilled water, and ethanol, air dry. Use dedicated sponges for each treatment.

2.

Using

a

kimwipe

tissue,

coat

the

inner

side

of

notched

or

offset

plate

with

acrylease

(Stratagene)

and

allow

to

dry--I

do

not

treat

the

top

2

inches

of

the

plate

since

I

feel

that

the

nonstick coating promotes leaking between wells behind the teeth of the comb. Buff well with a

kimwipe soaked in ethanol for a clean finish; this takes some elbow grease to get the streaks off of

the plate. Change gloves before working with bind silane and take care not to cross contaminate

the plates with the two treatments. The acrylease treatment only needs to be repeated every four

gels or so.

3.

Prepare

fresh

1

ml

binding

solution

by

making

a

solution

0.0005-0.001%

bind

silane

(Sigma #M-6514) in 95% ethanol, 0.5% glacial acetic acid. Apply with a kimwipe and coat the

inner

side

of

the

larger

plate

with

one

ml

and

allow

to

dry

4-5

minutes.

Wipe

the

plate

with

;.