Latest revision as of 23:55, 27 October 2012

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Protocols

How to Analyze 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 xμL (see Large Canvas AFM Protocol and Small Canvas AFM Protocol) of sample
Add yμL (see Large Canvas AFM Protocol and Small Canvas AFM Protocol) of 1xTE Buffer
Add zμL (see Large Canvas AFM Protocol and Small Canvas AFM Protocol) 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: 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

Select compute melt
Set 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

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

How to Make Small SST Canvas

Strand Mixture

Mix 1 μM solution of D-DNA SSTs from even rows (30 SSTs)
Mix 1 μM solution of D-DNA SSTs from protector strands (12 strands)
Mix 1 μM solution of D-DNA SSTs from rows 3, 5, 7, and 9 for each handle variation

Adding the L-DNA

Add L-DNA strand A to canvas solution in a 1:7 ratio
Add L-DNA strand B to purified solution in a 1:7 ratio

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

How to Make DNA Origami

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

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

2% aqueous uranyl formate stain solution (3 mL H20, 0.06 g uranyl formate):

- Uranyl formate (EMS, powder, store with parafilm over lid because seems to degrade in air)
- Weigh out 0.06 g uranyl formate in 10 mL beaker, using the balance in the hood
- Put in magnetic stir bar
- Put on magnetic stirrer under large inverted foil-topped beaker (don't stir it yet)
- Using a pipette, measure out 3 g H20 + 40 uL H20 = 3 mL H20 + 40 uL H20 (nuclease free, Ambion) in another 10 mL beaker
- Boil this water on a hot plate (NOT the same stirrer used for the uranyl formate) and shut off heat right when it boils
- Can carry over to the formate solution on the stirrer with gloves even though the water beaker is hot
- Pour hot water into formate beaker and turn on magnetic stirring; cover with large inverted foil-topped beaker

Glow discharging the grids:

- Formvar/Carbon coated grids (SPI # 3440C)
- Grab glass slide (e.g., VWR micro slides), gently wrap a piece of parafilm (e.g., 1.5 squares long, 1 sq wide) around a section of the glass slide
- Using tiny tweezers, grab grids from the edge and transfer to the parafilm wrapping on the slide
- Tweezers are Dumont N4AC from EMS. Buy your own tweezers; they break easily. Have one tweezer per grid if doing multiple grid preps in parallel.
- Put in EMS glow discharger. Settings: 25 mA, 45 s, 0.1 mBar, negative HT polarity. Press "start".

Putting sample on grid and staining:

- Materials: 15 mL falcon tube (wrap in foil), filter (Acrodisk, 0.2 um) that mounts on 5 mL syringe tip, 5 mL syringe (BD)
- Draw stain solution into the syringe, screw on filter, discharge through filter into fresh 15 mL tube
- Add 1 mL of uranyl formate solution into a fresh 2 mL eppendorf tube using a pipette
- Add 5 uL of 5N NaOH (JT Baker - this is DANGEROUS - DO NOT GET INTO EYES) into the 1 mL of stain solution in the eppendorf
- Vortex; the solution should get slightly darker
- Use a grid mat in a petri dish
- Line up tweezers, 1 tweezer per grid
- Grab grid edge and let rest on tweezers, suspended over air
- Pipette 3.5 uL of sample onto the grid for 4 min
- Take a piece of whatman paper, bend it in the middle
- Wick off sample by bringing whatman paper in contact with the grid edge from the side
- Immediately add 3.5 uL of stain solution onto the grid and let sit for 1 min
- Wick off the stain as before
- Leave for a minute or two to dry
- Transfer grid to mat at an angle: As grid nears mat surface, open tweezers slightly--the grid will still stick to the tweezers, and drag up to a ridge on the mat, at which point the grid will detach from the tweezers and rest up against the ridge on the mat
- It is crucial not to bend the grid or exert any forces on it
Put stuff that contacted uranyl formate in the radioactive waste bin

How to Make Large SST Canvas

Strand Mixture

Solution A: Mix 5 μL of exterior SSTs and interior SSTs from even rows (340 SSTs)
Solution B: Mix 5 μL of interior SSTs from odd rows (35 SSTs)
For 200 nM reaction, combine:
- 68 &mu:M solution A
- 7 &mu:M solution B
- 10 &mu:M 10x 12.5 mM folding buffer
- 15 &mu:M ddH₂O

Adding L-DNA

Add L-DNA strand A to canvas solution in a 1:7 ratio
Add L-DNA strand B to purified solution in a 1:7 ratio

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

@@ Line 68: / Line 68: @@
 *Select "Tapping Mode" in Fluid” and load settings
 *Use tape to remove top layer of mica disk
-*Add x&mu;L (see [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Large_Canvas_AFM_Specific_Notes Large Canvas AFM Protocol] and [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Small_Canvas_AFM_Specific_Notesl Small Canvas AFM Protocol]) of sample
+*Add x&mu;L (see [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Large_Canvas_AFM_Specific_Notes Large Canvas AFM Protocol] and [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Small_Canvas_AFM_Specific_Notes Small Canvas AFM Protocol]) of sample
 *Add y&mu;L (see [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Large_Canvas_AFM_Specific_Notes Large Canvas AFM Protocol] and [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Small_Canvas_AFM_Specific_Notes Small Canvas AFM Protocol]) of 1xTE Buffer
 *Add z&mu;L (see [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Large_Canvas_AFM_Specific_Notes Large Canvas AFM Protocol] and [http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Small_Canvas_AFM_Specific_Notes Small Canvas AFM Protocol]) of nickel solution if necessary

Biomod/2012/Harvard/BioDesign/protocols: Difference between revisions

Latest revision as of 23:55, 27 October 2012

Contents

How to Analyze DNA Structures

Making a Gel

Running a Gel

Imaging a Gel

Gel Purification

Preparing Mica for AFM

General AFM Protocol

How to Make L-DNA Layer

Dynamic Workbench: DD

NuPack

Oligo Analyzer

Sequence Massager

M-Fold

Annealing onto Template

How to Make Small SST Canvas

Strand Mixture

Adding the L-DNA

Annealing Template

Small Canvas AFM Specific Notes

How to Make DNA Origami

Strand Mixture (50 uL)

Adding the L-DNA

Annealing

TEM

How to Make Large SST Canvas

Strand Mixture

Adding L-DNA

Annealing Template

Large Canvas AFM Specific Notes

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