1

Version Space 의 실험적 고찰 III

2002.7.19

H M . Jang

2

Production Bead - probe

activation

coupling

blocking

coupling efficiency: 12 – 20%

3

Activation

15 A aliphatic arm

give flexibility

loading 75~125nmole / mg MPG

4

Separation

5

Procedure 전체 가설공간 생성 5’ end modified oligonucleotide Bead activation Coupling Blocking Separation PCR Photometric – assay

6

전체 가설공간 생성

cs

cs’

facultyfaculty’

four

four’

ee

ee’

staff

staff’

five

five’

?dept

?dept’

?status

?status’

?floor

?floor’

1. 95oC 5min denaturation

2. Temp decrease 1oc/1 cycle

3. ligation

4. 3% native gel

100bp

75bp

50bp

25bp

7

5’ end modified oligonucleotide

Amino linker C6 at 5’ end ; 20mer Concentration to 200pmol

by ethanol precipitation cs/ee/…..total 9 oligonucleotide

8

Bead activation

1. 1mg MPG uptake

2. add 6.4ul activation sol

3. remove supernatant

4. add 50ul coupling agent

5. washing 4 times

9

Coupling

1. add 5’ end modified oligo 100ul

2. 10ul reducing agent

3. incubation overnight at low

speed rotator

10

Blocking

1. 450ul blocking agent

2. gently vortex

3. incubation 2 hour

4. remove supernatant

5. washing 4 times

11

Separation

12

1st. Bead separation exp.

used oligo : EE / STAFF / 5 expected template :

EE-STAFF-5

1. 95oC 5min denaturation

2. 95oC 30sec denaturation

3. 58oC 2min annealing

4. 72oC 1min extention

13

PcR / electrophoresis

100bp75bp

50bp

25bp

EE / 5 EE / 4 CS / 4

template 2ul

primer 1u * 2

tag 1ul

14

Photometric assay

ee separation ee/staff separation ee/staff/5 separation 후 각 waste 의

OD 값을 260nm 에서 측정 이론적 비 = 18 : 6 : 0 측정치 = 4.0 : 0.107 : N/A

15

2nd. Bead separation exp.

used oligo : CS / FACULTY / 4 don’t DEPT/STAT/ FLOOR expected template :

1. 95oC 5min denaturation

2. 95oC 30sec denaturation

3. 58oC 2min annealing

4. 72oC 1min extention

CS FACULTY 4

CS FACULTY FLOOR

DEPT FACULTY 4

DEPT FACULTY FLOOR

CS STAT 4

CS STAT FLOOR

DEPT STAT 4

DEPT STAT FLOOR

16

Hot washing

55 oC hot water

17

PCR / electrophoresis

100bp

75bp

50bp

25bp

CS / 4DEPT / 4

DEPT /FLOOR

CS / 4

STAT / 4

STAT / FLOOR

CS / FLOOR

EE / 5

CS /5

DEPT /5

EE /4

EE/ FLOOR

N

A

G

A

T

I

V

E

18

Photometric assay 동일한 수의 가설이 만들어졌다고 전제 . CS / DEPT separation FACULTY / STAT separation 4 / FLOOR 후 각 waste 의 OD 값을 260nm 에서 측정

TOTAL

CS

/

DEPT

FACULTY

/

STAT

4

/

FLOOR

27 18 12 8

WASTE 9 6 4

O.D 4.000 0.922 0.682

AFTER 1 DAY 4.000 0.541 0.151

19

Discussion

primer – stick end region attach chromophore such as cy3, cy5 bead oriented problem

20

3st. Bead separation exp.

used oligo : CS / FACULTY / 4

don’t DEPT/STAT/ FLOOR used new primer : 15bp non stick end region

1. 95oC 5min denaturation

2. 95oC 30sec denaturation

3. 58oC 2min annealing

4. 72oC 1min extention

cscs’

facultyfaculty’

fourfour’

eeee’

staffstaff’

fivefive’

?dept

?dept’?status

?status’?floor

?floor’

21

PCR / electrophoresis

500bp

300bp

200bp

125bp

DEPT / 4

EE /4

CS / 4

DEPT / FLOOR

CS / FLOOR

EE / 5

DEPT /5

EE/ FLOOR

N

A

G

A

T

I

V

E

CS / 5

350bp

22

Discussion

Contamination Need more bead & oligo Other methods

Streptavidin – biotin pair Fluorescence conjugated primer set

23

Streptavidin – biotin pair