Biomod/2012/Harvard/BioDesign/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

• Run NanoScope 8.1 software
• Select "Tapping Mode" in Fluid” and load settings
• Use tape to remove top layer of mica disk
• Add x (see [Large Canvas AFM Protocol] and [Small Canvas AFM Protocol]) μL sample
• Add y (see [Large Canvas AFM Protocol] and [Small Canvas AFM Protocol]) μL of 1xTE Buffer
• Add z (see [Large Canvas AFM Protocol] and [Small Canvas AFM Protocol]) μL of nickel solution if necessary

• 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

• 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

MFold

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

Annealing Template

‘’’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

• Add 2.5 uL of 10uM first ribbon of L-DNA
• Anneal Ribbon A - See [Annealing onto Template]

• Purify - [Gel Purification]

• Anneal Ribbon B - [Annealing onto Template]

Annealing

‘’’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

Large Canvas SST Specifics

Annealing Template

‘’’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]