1. Erie Group Zimeng Li 2012-5-30 AFM real time visualization (7.01) A Fluid/Video AFM study of MMR (2.29) AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch Repair (5.30) ? Observation of Protein-DNA Interactions in real time (fall) Distinguish Proteins/Protein-DNA complex/EFM study Observation of Protein-DNA interaction involved in MMR Fluorescence-AFM Hybrid Imaging Distinguish Proteins/Protein-DNA complex/FRET study Ultramicroscopy AFM visualization of MutS Sliding Clamp Formation in DNA Mismatch Repair

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

7. Atomic Force Microscopy

8. From Force to Distance

9. AFM

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

12. Results

13. Recall

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

15. NiCl 2 No APTES, No Rinse, No Air Dry

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

17. Substrate/Support

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

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

20. Finally Solved Contamination

21. WaterLoSHiSNoS DryFluidDryFluidDryFluidDryFluid Decent Image29% (6/21) 18% (2/11) 50% (1/2) 100% (1/1) Great Image38% (8/21) 18% (2/11) 13% (1/8) Total67%36%50%13%0%(1)100% Success ‘Probability’ Tip consumption: ~70, 30% gets DNA, 30% gets something real, 30% bad

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

23. Resolution Improvement? AirWater

24. Tip is ‘hit’ so easily HeightPhase

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

26. 512, 2512,4256,6 256,10256,3

27. 96,396,10200,10 200,20512,20 Conclusion: 150nm scan 10s: 100,10 20s: 200,10

28. 512,401024,20512,20 100,10 Conclusion: 300nm scan 25s: 512,20 10s:100,10

29. 512,20256,20256,40 Conclusion: 100nm scan 12s: 256,20 6s: 256,40

30. 256,5256,1096,10 512,10512/256,20 Conclusion: 1um scan 50s:512,10;256,5 25s:256,10

31. Old Tips – avoid it

32. Injection

33. As time goes up, dirty things come out 10:11:33pm8:09:06pm

34. Injection of MutS Before After

35. How is DNA bound to mica? Competition between Mg 2+ and Na + Working with salt buffer

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

37. 512,5

38. 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 Hi-salt buffer

39. Hi-salt buffer injection Before After

40. After evaporation Before After

41. After evaporation

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

44. WaterLoSHiSNoS DryFluidDryFluidDryFluidDryFluid Decent Image29% (6/21) 18% (2/11) 50% (1/2) 100% (1/1) Great Image38% (8/21) 18% (2/11) 13% (1/8) Total67%36%50%13%0%(1)100% Success ‘Probability’ Tip consumption: ~70, 30% gets DNA, 30% gets something real, 30% bad

45. Great imageDecent image New tips31 Old tips12 My operations – wrong?

46. 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 Current Protocol

47. 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? To-dos