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this annealing run was dialyzed before imaging through a 25nm pore membrane against 1x TAE buffer (30 mins) PowerPoint Presentation
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this annealing run was dialyzed before imaging through a 25nm pore membrane against 1x TAE buffer (30 mins)

this annealing run was dialyzed before imaging through a 25nm pore membrane against 1x TAE buffer (30 mins)

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this annealing run was dialyzed before imaging through a 25nm pore membrane against 1x TAE buffer (30 mins)

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  1. D. Neff, expts done 7-6-2011This report shows 2rh window origami (two rhodamine labeled staples/origami). Massud annealed and stored this origami at 4 deg. C for ~ 1 week before dialysis and deposition shown here.

  2. this annealing run was dialyzed before imaging through a 25nm pore membrane against 1x TAE buffer (30 mins) • I am unsure of the staple:plasmid:rhodamine ratios used in origami synthesis • all images taken with the ixon3 (photon count capable) CCD and TM AFM • AFM and fluor. images are of the very same coverslip, AFM was NOT on fluor microscope stage • for spot analysis 9 pixels per spot (a 3x3 pixel area around the spot center) were averaged each frame • background plots look smoother because many more pixels were averaged than for spot (point source) analysis

  3. Preparing 2-rhodamine labeled DNA origami sample for fluorescence microscope (Dawn's original) • Clean glass coverslip • Make light scratch on one side of glass coverslip with diamond scribe • Sonicate in ethanol for 15 minutes • Rinse with UV-treated ddH2O and dry with N2 • Irradiate with UV light for 10 minutes • Place 1 µL of 2-rhodamine labeled origami and 9µL UV-treated ddH2O on coverslip (may have to adjust amount of origami depending on concentration but make sure you have a total of 10µL of liquid) • Let stand at room temperature for 10 minutes • Rinse coverslip with UV-treated ddH2O and dry with N2 • Image using fluorescence microscope. • Filter set 2 • 100x oil objective • 100msec exposure time and EM gain of 2500 for the Roleramgi camera • Preparing 2-rhodamine labeled DNA origami sample for fluorescence microscope (Neff proceedure 7-6-2011) • Clean glass coverslip • Make light scratch on one side of glass coverslip with diamond scribeSonicate in ethanol for 15 minutes • Rinse with UV-treated ddH2O and dry with N2 • Irradiate with UV light for 10 minutes • Place 1 µL of 2-rhodamine labeled origami and 9µL UV-treated ddH2O on coverslip (may have to adjust amount of origami depending on concentration but make sure you have a total of 10µL of liquid) • Let stand at room temperature for 10 minutes • Rinse coverslip one time with UV-treated ddH2O and dry with N2 • Image using fluorescence microscope. • Filter set 2 ex max 560, em max 630 - Hg lamp • 100x oil objective pixel = 129nm • imaging params with ixon3 are indicated beside images

  4. display 1500-4500 counts of 65536 total 1090 um2 48 point sources so here there are ~1 origami / 23um2 Filename: 150ms-50em-256x256center.sif Date and Time: Wed Jul 06 16:48:46 2011 Software Version: 4.18.30004.0 Temperature (C): -75 Model: DU897_BV Data Type: Counts Acquisition Mode: Frame Transfer Trigger Mode: Internal Exposure Time (secs): 0.15 Cycle Time (secs): 0.15027 Frequency (Hz): 6.6547 Frame Transfer Mode: Kinetics Number in Series: 1200 Readout Mode: Image {left,right,bottom,top}: 128,383,128,383 Horizontal Binning: 1 Vertical Binning: 1 Horizontally flipped: false Vertically flipped: false Clockwise rotation: false Anti-Clockwise rotation: false Vertical Shift Speed (usecs): 0.5 Pixel Readout Rate (MHz): 10 Baseline offset: 0 Number of prescans: 0 Baseline Clamp: ON Clock Amplitude: Normal Output Amplifier: Electron Multiplying EM Gain level: 50 Serial Number: 5762 Pre-Amplifier Gain: 5 SR163: Wavelength (nm): 500 Grating Groove Density (l/mm):200 Count Convert Mode: Counts Spurious Noise Filter Mode: No Filter Photon counted: false Data Averaging Filter Mode: No Filter 33 um time .150 seconds 33 um time 180 seconds 33 um

  5. plots for spots indicated in last slide (150ms-50em-256x256center.tif)

  6. first 75 seconds of same plot as last slide (150ms-50em-256x256center.tif)

  7. display 0-1500 photons of 65536 1 3 Filename: 120ms-100em.tif Date and Time: Wed Jul 06 16:42:25 2011 Software Version: 4.18.30004.0 Temperature (C): -75 Model: DU897_BV Data Type: Not available Acquisition Mode: Frame Transfer Trigger Mode: Internal Exposure Time (secs): 0.12 Cycle Time (secs): 0.12027 Frequency (Hz): 8.3146 Frame Transfer Mode: Kinetics Number in Series: 330 Readout Mode: Full Resolution Image Horizontal Binning: 1 Vertical Binning: 1 Horizontally flipped: false Vertically flipped: false Clockwise rotation: false Anti-Clockwise rotation: false Vertical Shift Speed (usecs): 0.5 Pixel Readout Rate (MHz): 10 Baseline offset: 0 Number of prescans: 0 Baseline Clamp: ON Clock Amplitude: Normal Output Amplifier: Electron Multiplying EM Gain level: 100 Serial Number: 5762 Pre-Amplifier Gain: 5 SR163: Wavelength (nm): 500 Grating Groove Density (l/mm):200 Count Convert Mode: Photons Sensitivity: 12.08 Detection Wavelength (nm): 630 Spurious Noise Filter Mode: No Filter Photon counted: false Data Averaging Filter Mode: No Filter 16 um background 2 time .120 seconds total 384 um2 13 point sources so here there are ~1 origami / 30um2 16 um time 40 seconds 24 um

  8. plots for spots indicated in last slide (120ms-100em.tif)

  9. AFM of same coverslip as preceeding slides total area in these 3 scans; 49 um2 +36um2 + 64 um2=149um2 total # origami in this area = 11 so we see about one origami / 15um2