1 Version Space 의 실험적 고찰 III 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 ’ faculty faculty ’ four four ’ ee ee ’ staff staff ’ five five ’ ? dept ? dept ’ ? status ? status ’ ? floor ? floor ’ 1.95 o C 5min denaturation 2.Temp decrease 1 o c/1 cycle 3.ligation 4.3% native gel 100bp 75bp 50bp 25bp

7 5’ end modified oligonucleotide Amino linker C 6 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 1 st. Bead separation exp. used oligo : EE / STAFF / 5 expected template : EE-STAFF o C 5min denaturation 2.95 o C 30sec denaturation 3.58 o C 2min annealing 4.72 o C 1min extention

13 PcR / electrophoresis 100bp 75bp 50bp 25bp EE / 5EE / 4CS / 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 : : N/A

15 2 nd. Bead separation exp. used oligo : CS / FACULTY / 4 don’t DEPT/STAT/ FLOOR expected template : 1.95 o C 5min denaturation 2.95 o C 30sec denaturation 3.58 o C 2min annealing 4.72 o C 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 o C hot water

17 PCR / electrophoresis 100bp 75bp 50bp 25bp CS / 4 DEPT / 4 DEPT /FLOOR CS / 4 STAT / 4 STAT / FLOOR CS / FLOOR EE / 5 CS /5 DEPT /5 EE /4 EE/ FLOOR NAGATIVENAGATIVE

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 WASTE964 O.D AFTER 1 DAY

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

20 3 st. Bead separation exp. used oligo : CS / FACULTY / 4 don’t DEPT/STAT/ FLOOR used new primer : 15bp non stick end region 1.95 o C 5min denaturation 2.95 o C 30sec denaturation 3.58 o C 2min annealing 4.72 o C 1min extention cs cs ’ faculty faculty ’ four four ’ ee ee ’ staff staff ’ five five ’ ? dept ? dept ’ ? status ? status ’ ? floor ? floor ’

21 PCR / electrophoresis 500bp 300bp 200bp 125bp DEPT / 4 EE /4 CS / 4 DEPT / FLOOR CS / FLOO R EE / 5 DEPT /5 EE/ FLOOR NAGATIVENAGATIVE CS / 5 350bp

22 Discussion Contamination Need more bead & oligo Other methods Streptavidin – biotin pair Fluorescence conjugated primer set

23 Streptavidin – biotin pair