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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

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

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DNA Mismatch Repair

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Sliding Clamp Formation Tessmer, et.al (2008)

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Sliding Clamp Formation Qiu, et.al (2012)

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

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Atomic Force Microscopy

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From Force to Distance

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AFM

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

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Results

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Recall

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

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NiCl 2 No APTES, No Rinse, No Air Dry

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

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Substrate/Support

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New Substrate Construct RMS: 220pm RMS: 80 pm

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

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Finally Solved Contamination

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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

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

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Resolution Improvement? AirWater

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Tip is hit so easily HeightPhase

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

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

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

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

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

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

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Old Tips avoid it

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Injection

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

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Injection of MutS Before After

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

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Hi-salt buffer Direct fluid imaging, 512,2.3

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512,5

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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

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Hi-salt buffer injection Before After

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After evaporation Before After

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After evaporation

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Lo-salt Buffer 9:45:20pm 9:55:27pm Indirect/direct fluid image: doesnt move

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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

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Great imageDecent image New tips31 Old tips12 My operations wrong?

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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

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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