Biomod/2011/Harvard/HarvarDNAnos:Tutorials

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

A Guide to AFM Image Artifacts
PDF by Paul West and Natalia Starostina

Disulfide Formation from Thiols
According to the Jeremy Sanders Group (http://www-sanders.ch.cam.ac.uk/disulfides.htm):



Dynamic Light Scattering

 * Wikipedia article on Dynamic Light Scattering
 * The DLS measures particle diffusion D and uses the Stokes-Einstein Formula to determine radius R:
 * $$D = \frac{k_\mathrm{B} T}{6\pi\,\eta\,R}$$


 * 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 $$\mu$$ is the inverse of the drag coefficient $$\zeta$$:
 * $$\zeta = 6 \pi \, \eta \, R,$$


 * Reference on DLS of nanoparticles
 * Another reference on DLS
 * The DLS can also measure the Zeta potential of the nanoparticles

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.

Equilibrium
Nupack will do multistrand 2ary structure analysis to get concentrations for each strand-strand complex in equilibrium.

Kinetics
Here are some tricks for kinetics modeling. These are all based on material from Dave Zhang.

1) the hybridization on rate is approximately k_on = 10^-7 /M/s

so a fixed A-A' gets hit by Z molecules at a rate (10^-7)*[Z] per second, where [Z] is the concentration of Z molecules in Molar.

2) if you can get a free energy difference between 2 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

then the off-rate is 1/t_off = (10^-7/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

3) 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 ~ sqrt(B) * (12 microseconds)

So you can compare this strand-displacement time t with the collision time 1/k_on and the dissociation time t_off to see if you're diffusion limited. If you are, things become easier to model: if there is enough toehold so that z always sticks on and ultimately displaces a' then the collision rate is ~ your rxn rate. If there is only a probability p of going to completion following a collision then it should be ~ p*(collision rate).

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. Then 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?
 * How much free energy is released due to entropy when a 5 nm gold particle escapes from a 40 nm box to explore the full volume of the test tube? How much pressure does it exert on the box walls? How much pressure is the DNA capable of exerting to keep it in?!
 * What is Boltzmann probability of box opening with 1, 2 or 3 latches?
 * How many times per second do 2 DNA strands collide as a fxn of concentration?

 