Lab 2 Practical Flashcards

plasmid

bacterial mini chromosome or vector

cloning

capturing a piece of foreign DNA into bacterial plasmid, then replicating that plasmid in bacteria

heterologous protein expression

transcription of a foreign gene within bacterial cells

Why do we want to purify proteins after expressing them in bacteria?

to generate antibodies and to study biochemical properties of a protein.

why express proteins in bacteria?

Proteins are hard to obtain from an organism’s own cells, which might be scarce in the first place. bacteria are cheap and easy to grow.

agarose gel electrophoresis uses

helps us visualize the presence, and size, of DNA in a sample. Separates DNA based on size and electrical charge. Helps us check if PCR succeeded.

why do we use agarose

it is easy. agarose is a polysaccharide purified from agar, which itself is isolated from seaweed. made up of repeating units of a disaccharide. Agarose polymers can dissolve in buffer at high temperatures but aggregate with each other and hydrogen bond with each other to form a solid gel at room temperature. The polymers hydrogen bond with each other to first form double helical structures, which can then aggregate with each other to form the gel. Pore sizes can vary with concentration of agarose. serves as molecular sieves. More agarose = smaller pores.

gel polymerization, what do we use during this process?

we use a plastic gel comb to make small indentations called wells in our gel to hold the DNA. The gel is then placed in a gel box where it will be submerged in a running buffer that allows the generation of an electrical current that flows through the gel to separate he DNA fragments. (DNA’s phosphate backbone carries negative charge in slightly basic pH of the buffer. Thus, DNA migrates towards positive pole in electrophoresis).

markers/ladders

DNA fragments of known lengths are placed in separate distinct well and by comparing the migration distance of the unknown fragments with distance traveled by the marker fragments, one can determine the approximate size of the unknown fragments.

ethidium bromide

slides/intercalates in between the stacked base pairs of the double helix, which stains the DNA fragments so we can visualize them. The gel can be either submersed in ethidum bromide solution after completion of gel electrophoresis or just directly added to agarose before casting gel. Requires UV light to visualize, or if you have a lot of it you can visualize it as bright orange.

loading dye components

added to DNA fragments prior to electrophoresis. Contains TrisHCl buffer to maintain pH at slightly basic, the compound EDTA to help inactivate DNA destroying enzymes (DNases) by chelating divalent cations used as cofactors for many enzymes, glycerol/ficoll to sink the sample to the bottom of the well, and tracking dyes such as bromophenol blue or xylene cyan to color the sample so you can see it while loading. the gel. The dyes move through the gel at a set pace so yo Luca keep track of roughly how far your DNA has migrated through the gel even if you can’t see DNA directly. Also contains boric acid to provide ions for current.

gel electrophoresis purification

cut out slice of agarose from gel that contains the PCR band (try to get the least amount of agarose as possible), then put a chaotropic salt to disrupt the hydrogen bonds of the gel with heating, and this breaks down the agarose gel to release the DNA and keeps the gel liquid at room temperature (ex includes guanidinium chloride or sodium iodide. The DNA fragments in solution would be captured by silica resin that binds to DNA fragments. The slightly acidic pH of chaotropic salt solution helps DNA interact with the silica resin. Then the DNA can be washed and purified and eluted.

QG solution

yellow, contains chaotropic salts in high concentrations (guanidine thiocyanate). Decreases the interactions between water molecules and DNA to favor interaction between DNA and the silica resin and lowers pH to slightly acidic of 6.6.

isopropanol

affects solubility of DNA (makes it insoluble) and helps DNA stick to resin.

Wash buffer PE and empty spin through

contains ethanol, dry spin to get rid of residual traces of alcohol that are not removed to prevent contamination problems.

elution buffer

add straight to the silica resin (otherwise % recovery of DNA would be dramatically lower). contains low salt concentration and higher pH of 8.5 to interfere with the binding of DNA to silica and thus wash the DNA right off the silica to collect.

size of pore and resolution

Gels are usually 1%-3% and the lower percent, 1%, has resolve of 5000-100 bp (the sweet spot of the bp of the DNA you are trying to separate. Too small and DNA will fly out of the gel and too big and they won’t be able to fit through the pores and won’t separate.