Biomod/2012/Harvard/BioDesign/protocols
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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
- 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
Settings:
- Compute melt
- Concentration: 1 μM
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
- Click Reverse and Complement as needed
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
‘’’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
TEM
Large Canvas SST Specifics
Strand Mixture
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]
