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

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==How to Make==
==How to Make==
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
#Add 2.5 uL of 10uM ribbon "a" of an L-DNA design
#Place in a PCR machine under the 40 Down protocol (add details of thermocycler program here)
#Gel purify
#Add 2.5 uL of ribbon "b" of an L-DNA design
#Place in PCR machine under the 40 Down protocl
Gel Purification
#Gel
50 nM final concentration of template
Create 2 sets of tubes of each Mg++ concentration for 6 tubes total
2. 72HR
3. Gel purify


==How to Analyze==
==How to Analyze==

Revision as of 23:03, 26 October 2012

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Protocols


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)

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)


Small Canvas SST

How to Design

Small Canvas SST Sequence Files


In order to design and further manipulate the small canvas SST files, use the following tools:

DyNAMiC Workbench

DyNAMic Workbench is an online tool that we used for designing and manipulating our DNA sequences to anneal at specific temperatures.

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

NUPACK

NUPACK is an online tool that we used for computing the temperature at which our SST structures would melt.

Settings:

  • Compute melt
  • Concentration: 1 μM


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:

  • 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 Sequence Massager is an online tool that we used for reversing or finding the complement strands for our SST sequences.

  • Click Reverse and Complement as needed


MFold

MFold is an online tool that we used for determining the temperatures at which our SST structures would form.

Settings:

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

How to Make

Making 1µM D-DNA Strand Solution

  1. Stock: 66 unique 100 µM strands
  2. Add 5 µL of each D-DNA strand into a PCR tube
    1. Nothing left in Well F3
    2. Nothing left in Well C4
  3. Add 170 µL of DD H2O
  4. End: 500 µL of 66 unique 1 µM D-DNA strands

Making 160mM Mg Buffer

  1. Stock: 1M MgCl2
  2. Dilute to 160 mM MgCl2 -1.6 mL 1 M MgCl2 and 8.4 mL H2O
  3. End: 10 mL of 160mM Mg Buffer

Setting Up SST Reaction

  1. Add 20 µL of D-DNA strand solution to 20 µL of our diluted buffer
  2. Add 160 µL of H2O
  3. End: 200 µL solution of 100 nM of each D-DNA strand and 16 mM of MgCl2 buffer

Thermal Cycler

  1. Turn dial to big tube
  2. Files -> New
  3. Lid temperature set at 105 degrees
  4. Wait
  5. Select 44 degrees
  6. Set at 30 minutes
  7. Hold at 20 degrees
  8. Exit
  9. Save as “Hold44”
  10. Run
  11. Approximately 12:20PM-12:50PM
  12. It will read “Hold at 20 degrees” when done.


How to Analyze

DNA Origami

How to Design

  1. Download Cadnano2 and follow installation directions
  2. Watch the tutorials on YouTube
  3. Download and manipulate DNA Origami file and generate DNA staple sequences. Edit and add sequences as needed.
  4. Use DyNAMiC Workbench to generate sequences for any strands needed outside the scaffold
  5. Order strands via Integrated DNA Technologies or a similar DNA synthesis company

How to Make

Making Origami Template (50 uL)

  1. In a PCR tube, add 20 uL of 200 nM staples
  2. Add 12.5 uL of 200 nM p8064 scaffold
  3. Add 5 uL of 110 mM Mg++
  4. Add 7.5 uL ddH2O
  5. Place in a PCR machine under 72HR program (add details of the thermocycler program here)
  6. Gel purify

Adding the L-DNA

  1. Add 2.5 uL of 10uM ribbon "a" of an L-DNA design
  2. Place in a PCR machine under the 40 Down protocol (add details of thermocycler program here)
  3. Gel purify
  4. Add 2.5 uL of ribbon "b" of an L-DNA design
  5. Place in PCR machine under the 40 Down protocl

Gel Purification

  1. Gel

50 nM final concentration of template Create 2 sets of tubes of each Mg++ concentration for 6 tubes total 2. 72HR 3. Gel purify

How to Analyze

Large Canvas SST

How to Design

How to Make

How to Analyze

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