Biomod/2012/Harvard/BioDesign/protocols

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Protocols

Analyzing the DNA Structures

Making a Gel

  • Create gel mixture
    • 120mL 0.5x TBE buffer
    • 2.4g agarose (powder)
    • ~10mL extra H2O for correction of volume during evaporation
  • Heat up in microwave for 2 min at full power until agarose melts and solution boils
  • Wait ~5 min for the solution to cool down, swirl in water briefly to aid
  • Add 1mL of 1.2M MgCl2
  • Add 6μL of 10,000x SybrSafe stock solution
  • Pour into snug gel tray in gel box, and put in combs, let cool for >15 min
  • Use comb to scoop out bubbles in agarose as needed


Running a Gel

  • Turn gel sideways in gel box
  • Add .5xTBE with 10mM Mg buffer to the gel box until at fill line
  • Mix 5μL of sample to 1μL of loading dye and add to each well
  • Add 1μL of ladder to lanes on both ends
  • Add top onto tray so that red terminal is pointed towards you, black terminal is pointed away
  • Set at 90V
  • Set max current to 400 mA
  • Set max power 100 Watts
  • Add ice water to outside of gel tray if necessary
  • Run for 1.5 to 3 hours

Imaging a Gel

  • Take gel out and place on grid of scanner
  • Open Typhoon FLA 9000 icon, Fluorescence setting
  • Type in a filename and select destination folder
  • Choose: SYBR Safe Mode, 400V PMT, 100μm resolution, preset values for correction
  • Set pre-scan area
  • Adjust final scan area with pre-scan data
  • Scan
  • Adjust brightness
  • Remove gel
  • Clean machine with ethanol and water

Gel Purification

  • Transport gel to darkroom
  • Place gel on viewing surface
  • Wear UV protection glasses and view gel under UV
  • Cut out the glowing band and remove the piece
  • Lay it on its side and trim the band
  • Place gel band in labelled tube
  • Dispose of excess gel in waste container labeled specifically for gel waste
  • Use pestle to crush sample inside the tubes using the pointed end.
  • Spin crushed gel at 400 rcf for 30 seconds to get gel to bottom
  • Cut off tip and invert inside freeze and squeeze tube
  • Use tube cutter to cut off tip of tube with gel contained within.
  • Spin down gel at 400-1000 rcf for 4-5 minutes

Preparing Mica for AFM

  • Put 5-minute epoxy into small weighboat
  • Use large pipette to mix epoxy together
  • Add a small dot of epoxy to disk center
  • Place mica on disk
  • Evenly distribute epoxy below surface of mica by pushing down on it with a pipette

General AFM Protocol

  • Place mica in AFM
  • Place tip on cleaned fluid cell and secure with spring clip
  • Secure fluid cell
  • Move the mica up so that the tip is close to the surface
  • Algin laser with the tip, adjust to increase sum through the two laser knobs and mirror
  • Set vertical and horizontal offset closer to zero
  • Auto-tune, can adjust Q
  • Check 5k sweep frequency for clean peaks
  • Engage
  • Set scan size to 1nm, check amplitude setpoint, set offsets to 0, integral gain to 3 and 6
  • Change scan size to desired image size
  • Select capture directory and capture
  • Withdraw when done
  • Remove and clean fluid cell
  • Remove and clean mica

How to Make L-DNA Layer

Dynamic Workbench: DD

  • Login
  • Click Tools -> DD
  • Add sequences, and fix base positions - capital letters remain constant, lower case letters mutate (double click on sequence to edit)
  • Select desired nucleotides to include in mutations (double click on composition and choose from scroll down menu)
  • Hit mutate - the lower the score, the better

NuPack

Settings:

  • Compute melt
  • Concentration: 1 μM

Oligo Analyzer

Settings:

  • Target type: DNA
  • Oligo Conc: 1 μM
  • Na+ Conc: 0mM
  • Mg++ Conc: 10mM
  • dNTPs Conc: 0 mM
  • Use Analyze and Self-Dimer to optimize

Sequence Massager

  • Click Reverse and Complement as needed


M-Fold

Settings:

  • Na+: 0 mM
  • Mg++: 10 mM
  • Folding temperature: 25°C

Annealing onto Template

‘’’40°C Down Anneal’’’

Temperature Control Mode: Calculated

Lid Control Mode: Tracking at 5°C above

  • Incubate at 40.0°C for 20 minutes
  • Decrease by 1.0°C every cycle
  • Cycle to step 1 for 15 more times
  • Incubate at 4.0°C forever

Small Canvas SST Specifics

Strand Mixture

Adding the L-DNA

Annealing Template

PCR Machine: 17 Hour Anneal

Temperature Control Mode: Calculated

Lid Control Mode: Tracking at 5°C above

  • Incubate at 90.0°C for 10 minutes
  • Decrease by 1.0°C every cycle
  • Cycle to step 1 for 29 more times
  • Incubate at 60.0°C for 20 minutes
  • Decrease by 1.0°C every cycle
  • Cycle to step 3 for 35 more times
  • Incubate at 4.0°C forever

Small Canvas AFM Specific Notes

Use 5μL sample and 30μL 1x TE Buffer with 20μL nickel and follow [General AFM Protocol]

DNA Origami Specifics

Strand Mixture (50 uL)

  • In a PCR tube, add 20 uL of 200 nM staples
  • Add 12.5 uL of 200 nM p8064 scaffold
  • Add 5 uL of 110 mM Mg++
  • Add 7.5 uL ddH2O

Adding the L-DNA

Annealing

PCR Machine: 72 Hour Anneal

Temperature Control Mode: Calculated

Lid Control Mode: Tracking at 10°C above

  • Incubate at 80.0°C for 5 minutes
  • Decrease by 1.0°C every cycle
  • Cycle to step 1 for 15 more times
  • Incubate at 64.0°C for 1 hour 45 minutes
  • Decrease by 1.0°C every cycle
  • Cycle to step 3 for 40 more times
  • Incubate at 4.0°C forever

TEM

Large Canvas SST Specifics

Strand Mixture

Annealing Template

PCR Machine: 17 Hour Anneal

Temperature Control Mode: Calculated

Lid Control Mode: Tracking at 5°C above

  • Incubate at 90.0°C for 10 minutes
  • Decrease by 1.0°C every cycle
  • Cycle to step 1 for 29 more times
  • Incubate at 60.0°C for 20 minutes
  • Decrease by 1.0°C every cycle
  • Cycle to step 3 for 35 more times
  • Incubate at 4.0°C forever

Large Canvas AFM Specific Notes

Use 5μL sample and 15μL 1x TE Buffer with no nickel and follow [General AFM Protocol]

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