1 / 10

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

D. Neff, expts done 7-6-2011 This 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. .

fox
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related