1 version space 의 실험적 고찰 iii 2002.7.19 h m. jang
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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
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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
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5’ end modified oligonucleotide
Amino linker C6 at 5’ end ; 20mer Concentration to 200pmol
by ethanol precipitation cs/ee/…..total 9 oligonucleotide
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Bead activation
1. 1mg MPG uptake
2. add 6.4ul activation sol
3. remove supernatant
4. add 50ul coupling agent
5. washing 4 times
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Coupling
1. add 5’ end modified oligo 100ul
2. 10ul reducing agent
3. incubation overnight at low
speed rotator
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Blocking
1. 450ul blocking agent
2. gently vortex
3. incubation 2 hour
4. remove supernatant
5. washing 4 times
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Separation
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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
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PcR / electrophoresis
100bp75bp
50bp
25bp
EE / 5 EE / 4 CS / 4
template 2ul
primer 1u * 2
tag 1ul
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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
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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
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Hot washing
55 oC hot water
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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
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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
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Discussion
primer – stick end region attach chromophore such as cy3, cy5 bead oriented problem
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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’
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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
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Streptavidin – biotin pair
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