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AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch Repair

AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch Repair. Erie Group Zimeng Li 2012-5-30. Contents. DNA Mismatch Repair Sliding Clamp Formation AFM techniques in fluid Results Future To-dos. DNA Mismatch Repair. Sliding Clamp Formation. Tessmer , et.al (2008).

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AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch Repair

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  1. AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch Repair Erie Group Zimeng Li 2012-5-30

  2. Contents • DNA Mismatch Repair • Sliding Clamp Formation • AFM techniques in fluid • Results • Future To-dos

  3. DNA Mismatch Repair

  4. Sliding Clamp Formation Tessmer, et.al (2008)

  5. Sliding Clamp Formation Qiu, et.al (2012)

  6. Techniques • Myosin V walk/Nano robot spider walk • Super resolution microscopy (Yildiz(2003);Nils Walter) • AFM (Kodera(2010);Nils Walter) • FRET (Qiu(2012)) • ?

  7. Atomic Force Microscopy

  8. From Force to Distance

  9. AFM

  10. AFM

  11. Atomic Force Microscopy • Fluid AFM techniques • Difference between thermal tune and cantilever tune • Substrates, special tips/treatment

  12. Results

  13. Recall

  14. DNA • Sample Buffer • APTES? • Rinse? • In Air? • Image Buffer • Tip

  15. NiCl2 No APTES, No Rinse, No Air Dry

  16. Contamination • Buffer – filter (twice) • Water - filter • Cantilever holder RMS: 220pm RMS: 37pm

  17. Substrate/Support

  18. New Substrate Construct RMS: 220pm RMS: 80 pm

  19. Dirty Tips? DNA: 600pm, Background: 400pm, Dots: 6nm RMS: 500 pm RMS: 80 pm

  20. Finally Solved Contamination

  21. Success ‘Probability’ Tip consumption: ~70, 30% gets DNA, 30% gets something real, 30% bad

  22. Case Study • Resolution compare • Fast image capability • Buffer dependence • Injection • Evaluate operations

  23. Resolution Improvement? Air Water

  24. Tip is ‘hit’ so easily Phase Height

  25. Fast Imaging Capability • Scan speed, scan points, integral gain, scan size; most importantly, tip’s health • 12s, 25s,50s

  26. Conclusion: 150nm scan 10s: 100,10 20s: 200,10

  27. Conclusion: 300nm scan 25s: 512,20 10s:100,10

  28. Conclusion: 100nm scan 12s: 256,20 6s: 256,40

  29. Conclusion: 1um scan 50s:512,10;256,5 25s:256,10

  30. Old Tips – avoid it

  31. Injection

  32. As time goes up, dirty things come out 8:09:06pm 10:11:33pm

  33. Injection of MutS Before After

  34. Working with salt buffer • How is DNA bound to mica? • Competition between Mg2+ and Na+

  35. Hi-salt buffer Direct fluid imaging, 512,2.3

  36. 512,5

  37. Hi-salt buffer • Indirect fluid imaging (i.e. dry first) • Initial: 120uL water+DNA • Injection: 120uL Hi-salt ->’lo-salt’ mixture • Evaporate: 120uL water • Eventually: 120uL Hi-salt+DNA

  38. Hi-salt buffer injection Before After

  39. After evaporation Before After

  40. After evaporation

  41. After evaporation

  42. Lo-salt Buffer 9:45:20pm 9:55:27pm Indirect/direct fluid image: doesn’t move

  43. Success ‘Probability’ Tip consumption: ~70, 30% gets DNA, 30% gets something real, 30% bad

  44. My operations – wrong?

  45. Current Protocol • 15min • Running a full cleaning cycle • Deposition/No incubation • Rinse once (Twice results in no DNA?) • 15-30min/cycle • Image/No Good?/Change tips/Image/… • Average 2~3tips/sample

  46. To-dos • Continue working with lo-salt buffer • If not working, try no-salt buffer • Adding NaCl to increase DNA mobility • Hi-salt – using APTES treated mica to reduce mobility • Does MutS work in water or no-salt buffer?

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