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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, 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.

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Phosphorylation 전의 마지막 결과

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  1. 2% agarose 10% PAA gel (첫번째) 대상: HPP (0~6) lane 1: oligomer mixture lane 2: hybridization (70->37) lane 3: ligation (16도, 15hr, 1ml, 100ml) lane 4: marker lane 5: PCR ctrl (처음한거) lane 6: ligation 후 바로 PCR lane 7: 3을 alkaline 처리한것 lane 8: 3을 desalting 한것 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Phosphorylation 전의 마지막 결과

  2. 2% agarose 10% PAA gel (두번째) 대상: HPP (0~6) lane 1: marker lane 2: kination한 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 premix만 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Kination 후의 첫 결과

  3. 2% agarose 대상: HPP (0~6) lane 1: marker lane 2: kination한 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

  4. 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 1 2 3 4 5 6 7 8 9 10 5’-phosphate로 실험한 첫 결과 • annealing temp. = 50도 • polymerization = 30 sec

  5. 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’-phosphate로 실험한 첫 결과

  6. 03, 06 annealing temp. = 52도 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’-phosphate로 실험한 두번째 결과

  7. 04, 05, 06, 36 annealing temp. = 50도 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’-phosphate로 실험한 세번째 결과

  8. 10% PAA gel 대상:TSP (15, 03, 04, 05, 06, 36:S/A) 모두 ppt를 PCR lane 1, 2: marker lane 3: TSP15 (A) lane 4: TSP03 (A) lane 5: TSP04 (A) - 다시 ppt한것 lane 6: TSP06 (A) - 다시 ppt한것 lane 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 5’-phosphate로 실험한 네번째 결과

  9. 현재 가지고 있는 vertex들의 Tm vertex 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 pair 1~6  problem size = 6 1~5  problem size = 5 0~3  problem size = 4 0~3 vertex : 0, 1, 2, 3 edge : 01, 02, 12, 13, 23 ( 2) weight : 3 ( 3), 5, 7 Tm (avg) = 59 ℃ 1~6 vertex : 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 ℃ 새로이 시작..

  10. 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 TSP 1~6 결과 I 1 2 3 4 5 6 7

  11. 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) TSP 1~6 결과 II 1 2 3 4

  12. template 양이 영향이 있는듯. negative control은 negative하지 않다. 중간의 weight sequence가 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 TSP 1~6 결과 III 1 2 3 4 5 6 7

  13. 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) TSP 0~3 결과 1 2 3 4 5 6 7

  14. 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 TSP 0~6 결과 1 2 3 4 5 6

  15. Think!!! • Template 양 (Oligomer 양) • Negative control : Primer dimer? or not? : 80 ~ 100bp • Hybridization/ Ligation 여부? • New Problem New construction vs. Renovation ?? 비슷한 Tm, 또는 높은 Tm의 primer addition of edges removal of edges TGGE

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