Biomod/2012/Harvard/BioDesign/protocols

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(How to Analyze)
Line 1: Line 1:
-
{{Template:Biomod/2012/Harvard/BioDesign}}
+
=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
-
<font size="5">Protocols</font>
 
 +
==Running a Gel==
 +
*Turn gel sideways in gel box
 +
*Add .5xTBE with 10mM Mg buffer to the gel box until at fill line
 +
*Mix 5&mu;L of sample to 1&mu;L of loading dye and add to each well
 +
*Add 1&mu;L 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, 100&mu;m 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
-
Clarity: Is the project description well-written and easy to understand? Does it include the background and motivation of the project, '''methods''', results, and discussion? '''Are the figures easy to understand?''' (10 points)
+
==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
-
Transparency: Are all of the raw experimental data and source files easily accessible? '''Would it be straightforward to attempt to reproduce the team's results?''' (5 points)
+
==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 [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Large_Canvas_AFM_Protocol Large Canvas AFM Protocol]] and [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Small_Canvas_AFM_Protocol Small Canvas AFM Protocol]]) &mu;L sample
 +
*Add y (see [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Large_Canvas_AFM_Protocol Large Canvas AFM Protocol]] and [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Small_Canvas_AFM_Protocol Small Canvas AFM Protocol]]) &mu;L of 1xTE Buffer
 +
*Add z (see [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Large_Canvas_AFM_Protocol Large Canvas AFM Protocol]] and [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Small_Canvas_AFM_Protocol Small Canvas AFM Protocol]]) &mu;L 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=
-
=Small Canvas SST=
 
-
 
-
==How to Design==
 
-
 
-
'''[https://www.dropbox.com/sh/lahfbz85lqiumr0/Yg9EL0uf3u Small Canvas SST Sequence Files]'''
 
-
 
-
 
-
In order to design and further manipulate the small canvas SST files, use the following tools:
 
'''[http://107.22.192.99:3000/ DyNAMiC Workbench]'''
'''[http://107.22.192.99:3000/ DyNAMiC Workbench]'''
-
DyNAMic Workbench is an online tool that we used for designing and manipulating our DNA sequences to anneal at specific temperatures.
 
-
 
-
#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
 
-
[[Image: DyNAMiC Workbench.png]]
+
*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
'''[http://nupack.org/partition/new NUPACK]'''
'''[http://nupack.org/partition/new NUPACK]'''
-
 
-
NUPACK is an online tool that we used for computing the temperature at which our SST structures would melt.
 
Settings:
Settings:
*Compute melt
*Compute melt
*Concentration: 1 &#956;M
*Concentration: 1 &#956;M
-
 
-
[[Image: NUPACK.png]]
 
-
 
'''[http://www.idtdna.com/analyzer/applications/oligoanalyzer/ Oligo Analyzer]'''
'''[http://www.idtdna.com/analyzer/applications/oligoanalyzer/ Oligo Analyzer]'''
-
 
-
Oligo Analyzer is an online tool that we used for determining if any of our SST structures would bind complementary to themselves.
 
Settings:
Settings:
Line 58: Line 110:
*dNTPs Conc: 0 mM
*dNTPs Conc: 0 mM
*Use Analyze and Self-Dimer to optimize
*Use Analyze and Self-Dimer to optimize
-
 
-
[[Image: Oligo_Analyzer.png]]
 
-
 
'''[http://www.attotron.com/cybertory/analysis/seqMassager.htm Sequence Massager]'''
'''[http://www.attotron.com/cybertory/analysis/seqMassager.htm Sequence Massager]'''
-
Sequence Massager is an online tool that we used for reversing or finding the complement strands for our SST sequences.
 
-
 
-
[[Image: Sequence_Massager.png]]
 
*Click Reverse and Complement as needed
*Click Reverse and Complement as needed
Line 71: Line 117:
'''[http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form MFold]'''
'''[http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form MFold]'''
-
 
-
MFold is an online tool that we used for determining the temperatures at which our SST structures would form.
 
Settings:
Settings:
Line 78: Line 122:
*Mg++: 10 mM
*Mg++: 10 mM
*Folding temperature: 25&#176;C
*Folding temperature: 25&#176;C
-
[[Image: MFold.png]]
 
-
==How to Make==
+
==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
-
Making 1µM D-DNA Strand Solution
+
==Small Canvas AFM Specific Notes==
-
#Stock: 66 unique 100 µM strands
+
Use 5&mu;L sample and 30&mu;L 1x TE Buffer with 20&mu;L nickel and follow
-
#Add 5 µL of each D-DNA strand into a PCR tube
+
[[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#General_AFM_Protocol General AFM Protocol]]
-
##Nothing left in Well F3
+
-
##Nothing left in Well C4
+
=DNA Origami Specifics=
-
#Add 170 µL of DD H2O
+
-
#End: 500 µL of 66 unique 1 µM D-DNA strands
+
-
Making 160mM Mg Buffer
+
==Strand Mixture (50 uL)==
-
#Stock: 1M MgCl2
+
*In a PCR tube, add 20 uL of 200 nM staples
-
#Dilute to 160 mM MgCl2 -1.6 mL 1 M MgCl2 and 8.4 mL H2O
+
*Add 12.5 uL of 200 nM p8064 scaffold
-
#End: 10 mL of 160mM Mg Buffer
+
*Add 5 uL of 110 mM Mg++
 +
*Add 7.5 uL ddH2O
-
Setting Up SST Reaction
+
==Adding the L-DNA==
-
#Add 20 µL of D-DNA strand solution to 20 µL of our diluted buffer
+
*Add 2.5 uL of 10uM first ribbon of L-DNA
-
#Add 160 µL of H2O
+
*Anneal Ribbon A - See [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Annealing_onto_Template Annealing onto Template]]
-
#End: 200 µL solution of 100 nM of each D-DNA strand and 16 mM of MgCl2 buffer
+
-
Thermal Cycler
 
-
#Turn dial to big tube
 
-
#Files -> New
 
-
#Lid temperature set at 105 degrees
 
-
#Wait
 
-
#Select 44 degrees
 
-
#Set at 30 minutes
 
-
#Hold at 20 degrees
 
-
#Exit
 
-
#Save as “Hold44”
 
-
#Run
 
-
#Approximately 12:20PM-12:50PM
 
-
#It will read “Hold at 20 degrees” when done.
 
 +
*Purify - [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Gel_Purification Gel Purification]]
-
==How to Analyze==
+
*Anneal Ribbon B - [[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#Annealing_onto_Template Annealing onto Template]]
-
=DNA Origami=
+
==Annealing==
 +
‘’’72 Hour Anneal’’’
-
==How to Design==
+
Temperature Control Mode: Calculated
-
# Download [http://cadnano.org Cadnano2] and follow installation directions
+
-
# Watch the [http://www.youtube.com/watch?v=cwj-4Wj6PMc tutorials] on YouTube
+
-
# Download and manipulate [https://www.dropbox.com/s/3d1te9lsl1tvh8t/120717_0118_sqhl_template_v0.json DNA Origami file] and generate DNA staple sequences. Edit and add sequences as needed.
+
-
# Use [http://107.22.192.99:3000/ DyNAMiC Workbench] to generate sequences for any strands needed outside the scaffold
+
-
# Order strands via [http://www.idtdna.com/site Integrated DNA Technologies] or a similar DNA synthesis company
+
-
==How to Make==
+
Lid Control Mode: Tracking at 10°C above
-
Making Origami Template (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
+
-
#Place in a PCR machine under 72HR program (add details of the thermocycler program here)
+
-
#Gel purify
+
-
Adding the L-DNA
+
*Incubate at 80.0°C for 5 minutes
-
#Add 2.5 uL of 10uM ribbon "a" of an L-DNA design
+
  Decrease by 1.0°C every cycle
-
#Place in a PCR machine under the 40 Down protocol (add details of thermocycler program here)
+
*Cycle to step 1 for 15 more times
-
#Gel purify
+
*Incubate at 64.0°C for 1 hour 45 minutes
-
#Add 2.5 uL of ribbon "b" of an L-DNA design
+
  Decrease by 1.0°C every cycle
-
#Place in PCR machine under the 40 Down protocol
+
*Cycle to step 3 for 40 more times
 +
*Incubate at 4.0°C forever
-
==How to Analyze==
+
==TEM==
-
#Run a sawtooth gel, so as to compare the sizes of the structure before and after L-DNA addition
+
-
#Image structures with Atomic Force Microscopy (AFM).
+
=Large Canvas SST Specifics=
-
#Image structures with Transmission Electron Microscopy (TEM).
+
==Strand Mixture==
 +
==Annealing Template==
 +
‘’’17 Hour Anneal’’’
-
=Large Canvas SST=
+
Temperature Control Mode: Calculated
-
==How to Design==
+
Lid Control Mode: Tracking at 5°C above
-
==How to Make==
+
*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
-
==How to Analyze==
+
==Large Canvas AFM Specific Notes==
 +
Use 5&mu;L sample and 15&mu;L 1x TE Buffer with no nickel and follow
 +
[[http://openwetware.org/wiki/Biomod/2012/Harvard/BioDesign/protocols#General_AFM_Protocol General AFM Protocol]]

Revision as of 02:30, 27 October 2012

Contents

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


  • 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

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


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]

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