Biomod/2011/Harvard/HarvarDNAnos:Tutorials

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At the Lab Bench



Different Types of Buffer in the Lab

  • Ingredients
    • EDTA = Ethylenediaminetetraacetic acid
      • Readily chelates divalent ions such as Mg2+
      • Useful for deactivating metal-dependent enzymes that can damage DNA or proteins
    • Tris = Tris(hydroxymethyl)aminomethane = (HOCH2)3CNH2
      • Extensively used buffer that has a pKa of 8 and a buffer range of pH = 7-9
    • Acetic Acid / Acetate (deprotonated acetic acid)
    • Boric Acid / Borate (deprotonated boric acid)
  • TAE Buffer - Tris-Acetate-EDTA
    • Used commonly for gel electrophoresis
    • Since borate inhibits the ligase enzyme, TAE is used instead of TBE Buffer when ligation (to a plasmid or other cloning vector, e.g.) is necessary after running the gel
  • TBE Buffer - Tris-Borate-EDTA
    • Also commonly used for gel electrophoresis
    • Produces more rigid gels than TAE, making the gel easier to handle and allowing you to run the gel faster (at higher voltages) without melting
    • Borate is an inhibitor for many enzymes, for better or worse
  • According to Adam, do not mix TAE and TBE buffer; for example, don't use TBE to make your gel and then TAE to run your gel; this will cancel out the buffering effect
  • LB Buffer - Lithium Borate
    • Lower conductivity, produces crisper resolution, and can be run at higher speeds (voltages) than gels made from TBE or TAE (because lower heat generation)
  • Folding Buffer consists of Tris, EDTA, MgCl2, and water
    • Ralf says that it is okay for folding buffer to contain acetate as well
    • Ralf also says that we should filter folding buffer every couple weeks to remove random dust particles, etc.


Different Types of Water in the Lab

  • diH2O - deionized water
    • Produced by filtering
    • Contains essentially no ions; has very low electrical conductivity and is very "hard"
    • May contain bacteria and other organic contaminants, although unlikely
    • Useful for cleaning because will suck up any surrounding ions
  • ddH2O - double distilled water
    • Produced by converting water from liquid to gas and then back to liquid; therefore, may contain volatile impurities
    • Contains some ions because ions are picked up during the condensation process; therefore, "softer" than DI water
    • Generally has less organic contamination than DI water
  • Both di and ddH2O are not good to drink, because they will leach ions and nutrients out of your body tissues
  • Nuclease-free, sterile water
    • Water in which microorganisms have been killed and which does not contain any nuclease (enzymes capable of cleaving the phosphodiester bonds between nucleotides)
    • May contain minerals and other impurities


Performing a Dilution (An Explanation of the "x" Notation)

  • The number before the "x" refers to the factor by which the reagent is too concentrated with respect to standard use. For example, when using 10,000x SYBR-Gold, one should dilute 5 uL of the SYBR-Gold in 45 mL of water to make the proper staining liquid (because 5 uL/(45+5) mL = 1/10,000). When using 10x folding buffer in a total reaction volume of 50 uL (this number includes the buffer), one should use 5 uL of the buffer.


Resuspending lyophillized strands to a defined concentration

  • Use the labels on the tube to resuspend to twice the desired concentration. Then measure the concentration with the nanodrop using the extinction coefficient of the sequence (use online calculator or tube label). Then calculate the dilution to get you to the exact desired concentration. Or do 4x, then 2x, then 1x etc.


Laboratory Equipment

Transmission Electron Microscope (TEM)


Atomic Force Microscope (AFM)

PDF by Paul West and Natalia Starostina on Image Artifacts

Dynamic Light Scattering Device (DLS)

D = \frac{k_\mathrm{B} T}{6\pi\,\eta\,R}
  • The Stokes-Einstein Formula derives from
  • the Einstein Relation:
Image:Screen shot 2011-06-20 at 7.31.25 PM.png
  • via Stokes' Law, because at low convection, mobility μ is the inverse of the drag coefficient ζ:
\zeta = 6 \pi \, \eta \, R,


Typhoon Gel Imager


Thermocycler


Centrifuge


Slow Shaker



Other

Disulfide Formation from Thiols

According to the Jeremy Sanders Group (http://www-sanders.ch.cam.ac.uk/disulfides.htm): Image:Screen_Shot_2011-07-25_at_10.57.41_AM.png

  • Two chemical reactions are used to generate the DCL: thiol oxidation, which generates a mixture of disulfides from thiols, and thiol-disulfide exchange, which allows a mixture of disulfides to exchange and reach equilibrium, so long as a catalytic amount of thiolate anion is present. These reactions occur spontaneously in aqueous solution at pH values between 7 and 9. Oxygen from the air is sufficient to oxidise the thiols, and so no special reagents are required. The reactions may be quenched by simply lowering the pH of the DCL.


Tricks for modeling/estimating DNA hybridization

Equilibrium

  • Nupack will do multistrand 2ary structure analysis to get concentrations for each strand-strand complex in equilibrium, as well as the probability-weighted ensemble of structures that each such strand-strand complex can adopt.


Kinetics

Here are some tricks for kinetics modeling of strand displacement processes, such as SD-based box opening. These are all based on material from Dave Zhang.

  • The hybridization on-rate constant is approximately k_on = between 10^6 /M/s and 10^7 /M/s depending on conditions, for a bi-molecular hybridization reaction
    • So a fixed A-A' gets hit by Z molecules at a rate (10^6)*[Z] per second, where [Z] is the concentration of Z molecules in Molar.
  • If you can get a free energy difference between two states using NuPack or an estimate, then e^(-deltaG/RT) is the equilibrium constant, K. K will have units of /M since K = [complex 1-2] / ([complex 1] [complex 2]) for an association reaction.
    • Note: RT at room temperature is about 0.6 kcal/mol
    • 1/K is the concentration of half-hybridization
    • The the off-rate is given by 1/t_off = (10^6/M/s) / K where t_off is the lifetime of the full complex
    • Using these formulas you can see that only after about 8 base pairs do you get something that is stable for tens of seconds and that hybridizes with high yield at 100 nM concentrations at room temperature.
  • Strand displacement: it's an unbiased 1D random walk with step-time of 12 microseconds
    • Thus the time taken to strand displace through B bases is t ~ (B^2) * (12 microseconds)
  • Toehold-mediated strand displacement kinetics saturates at a toehold length of around 7 bp


Modeling problems

  • Compare 1pN*1nm with k_b * T where k_b is boltzmanns constant and T is room temp in Kelvin. The former is the energy required to overcome a lock which exterts a 1 pN force over a 1 nm distance. The latter is the average energy due to thermal fluctuations in a system with 1 degree of freedom.
    • How do these compare to a kcal/mol?
    • How do they compare to the ~ 1 eV energies characteistic of covalent bonds?
    • How do they compare to the energy of base stacking + hydrogen bonding in a 10 bp duplex?
  • What is the Boltzmann probability of a box opening with 1, 2 or 3 DNA hybridization latches?
  • How many times per second do 2 DNA strands collide as a fxn of concentration?


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