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2% agarose 10% PAA gel ( 첫번째 ) 대상 : HPP (0~6) lane 1: oligomer mixture lane 2: hybridization (70->37) lane 3: ligation (16 도 , 15hr, 1 m l, 100 m l) lane 4: marker lane 5: PCR ctrl ( 처음한거 ) lane 6: ligation 후 바로 PCR lane 7: 3 을 alkaline 처리한것 lane 8: 3 을 desalting 한것. - PowerPoint PPT Presentation
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Phosphorylation 전의 마지막 결과
• 2% agarose
• 10% PAA gel ( 첫번째 )
• 대상 : HPP (0~6)lane 1: oligomer mixture
lane 2: hybridization (70->37)
lane 3: ligation (16 도 , 15hr, 1l, 100l)
lane 4: marker
lane 5: PCR ctrl ( 처음한거 )
lane 6: ligation 후 바로 PCR
lane 7: 3 을 alkaline 처리한것lane 8: 3 을 desalting 한것
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
Kination 후의 첫 결과
• 2% agarose
• 10% PAA gel ( 두번째 )
• 대상 : HPP (0~6)lane 1: marker
lane 2: kination 한 oligomer mixture
lane 3: oligomer mixture
lane 4: ligation 후 바로 PCR
lane 5: lig/ppt 하고 PCR
lane 6: PCR ctrl ( 새로한것 )
lane 7: 예전에 phosphate 없이 PCR했던것
lane 8: PCR premix 만
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
• 2% agarose
• 대상 : HPP (0~6)lane 1: marker
lane 2: kination 한 oligomer mixture
lane 3: oligomer mixture
lane 4: ligation 후 바로 PCR
lane 5: PCR ctrl
lane 6: 예전에 phosphate 없이 PCR했던것
1 2 3 4 5 6
5’-phosphate 로 실험한 첫 결과
• 10% PAA gel ( 세번째 )
• 대상 : HPP (0~6:S)
TSP (0~6:S/A)lane 1: marker
lane 2: oligomer mixture
lane 3: ligation result
lane 4: PCR ctrl ( 새로한것 )
lane 5: lig/ppt 하고 PCR
lane 6: ligation 후 바로 PCR
lane 7: PCR-TSP (S) - ppt
lane 8: PCR-TSP (S) - lig
lane 9: PCR-TSP (A) - ppt
lane 10: PCR-TSP (A) - lig
• annealing temp. = 50 도• polymerization = 30 sec
1 2 3 4 5 6 7 8 9 10
5’-phosphate 로 실험한 첫 결과
• 10% PAA gel ( 네번째 )
• 대상 : HPP (0~6:S)
TSP (0~6:S/A)lane 1: marker
lane 2: PCR-HPP (S) - ppt
lane 3: PCR-TSP (S) - ppt
lane 4: PCR-TSP (A) - ppt
lane 5: PCR ctrl ( 처음 한것 )
lane 6: oligomer mixture - TSP (S)
lane 7: ligation/ppt - TSP (S)
lane 8: oligomer mixture - TSP (A)
lane 9: ligation/ppt - TSP (A)
1 2 3 4 5 6 7 8 9
5’-phosphate 로 실험한 두번째 결과
• 03, 06
• annealing temp. = 52 도• polymerization = 30 sec
• 10% PAA gel ( 다섯번째 )• 대상 : TSP (0~3:S)lane 1: marker
lane 2: PCR- TSP03 (S) - ppt
lane 3: TSP03 (S) - lig/ppt
lane 4: PCR- TSP06 (S) - ppt
lane 5: TSP06 (S) - lig/ppt
lane 6~9: 예전에 했던 것들 .
1 2 3 4 5 6 7 8 9
5’-phosphate 로 실험한 세번째 결과
• 04, 05, 06, 36
• annealing temp. = 50 도
• 10% PAA gel ( 여섯번째 )• 대상 : TSP (03, 04, 05, 06,
36:S/A)lane 1: marker
lane 2: PCR- TSP03 (S) - ppt ( 지난번꺼 )
lane 3: PCR- TSP04 (A) -ppt
lane 4: PCR- TSP05 (A) - ppt
lane 5: PCR- TSP06 (A) - ppt
lane 6: PCR- TSP36 (S) - ppt
1 2 3 4 5 6
5’-phosphate 로 실험한 네번째 결과
• 10% PAA gel • 대상 : TSP (15, 03, 04, 05,
06, 36:S/A)• 모두 ppt 를 PCRlane 1, 2: marker
lane 3: TSP15 (A)
lane 4: TSP03 (A)
lane 5: TSP04 (A) - 다시 ppt 한것lane 6: TSP06 (A) - 다시 ppt 한것lane 7: TSP36 (A)
lane 8: TSP36 (A) - lig/ppt
lane 9: TSP15 (A) oligomer mixture
lane 10: TSP15 (A) - lig/ppt
1 2 3 4 5 6 7 8 9 10
새로이 시작 ..
• 현재 가지고 있는 vertex 들의 Tmvertex 0 = 57.6 ℃vertex 1 = 52.6 ℃vertex 2 = 57.3 ℃vertex 3 = 60.4 ℃vertex 4 = 49.4 ℃vertex 5 = 50.2 ℃vertex 6 = 53.6 ℃
• 적당하다고 생각되는 pair1~6 problem size = 61~5 problem size = 50~3 problem size = 4
• 0~3vertex : 0, 1, 2, 3edge : 01, 02, 12, 13, 23 ( 2)weight : 3 ( 3), 5, 7Tm (avg) = 59 ℃
• 1~6vertex : 1, 2, 3, 4, 5, 6 edge : 12, 13, 16, 23, 34, 35, 36, 45, 56 ( 2)weight : 3 ( 5), 7, 7, 11 ( 2)Tm (avg) = 53.1 ℃
TSP 1~6 결과 I
• Tm (avg) = 53.1℃
• 2.5% Agarose gel lane 1: marker
lane 2: 1X template (primer 100pmole)
lane 3: 0.1X “ (primer 50pmole)
lane 4: 0.02X “ (primer 100pmole)
lane 5: ligation
lane 6: 예전 TSP
lane 7: 예전 HPP
1 2 3 4 5 6 7
TSP 1~6 결과 II
• 2.5% Agarose gel lane 1: marker
lane 2: 0.02X template (primer 50pmole)
lane 3: 0.004X “ (primer 50pmole)
lane 4: 0.0008X “ (primer 100pmole)
1 2 3 4
TSP 1~6 결과 III
• template 양이 영향이 있는듯 .
• negative control 은 negative 하지 않다 .
• 중간의 weight sequence 가 문제가 있지는 않은듯 .
• 2% Agarose gel lane 1: marker
lane 2: 0.1X template (primer 25pmole)
lane 3: 0.01X “
lane 4: 0.001X “
lane 5: 0.0001X “
lane 6: negative control (only primers)
lane 7: ligation
1 2 3 4 5 6 7
TSP 0~3 결과
• Tm (avg) = 59℃
• 2% Agarose gel lane 1: ligation
lane 2: marker 0.1X template
lane 3: 0.1X template
lane 4: 0.01X “
lane 5: 0.001X “
lane 6: 0.0001X “
lane 7: negative control (only primers)
1 2 3 4 5 6 7
TSP 0~6 결과
• Tm (avg) = 55.6℃
• 2% Agarose gel lane 1: marker
lane 2: 1X template
lane 3: 0.1X “
lane 4: 0.01X “
lane 5: 0.001X “
lane 6: ligation
1 2 3 4 5 6
Think!!!
• Template 양 (Oligomer 양 )• Negative control : Primer dimer? or not?
: 80 ~ 100bp
• Hybridization/ Ligation 여부 ?
• New ProblemNew construction vs. Renovation ??
비슷한 Tm, 또는 높은 Tm 의 primer
addition of edges
removal of edges
TGGE
