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ButInTh33nd 04-13-10 08:25 PM

Studying for my Microbiology course
This thread is going to be nothing more than me going over my notes. Enjoy XD

Actually asking questions will probably help test me, so fire away. No guarantees I will actually answer them here though.

Okay, so first of all, we are working with super tiny amounts of material. In class, the most common unit we worked with were microlitres (uL), or 10^-6 L. In addition, 10^-9 is nanolitres, and 10^-12 is picolitres.

In terms of solutions, we referred to them as molarity and weight per volume. Molarity is mols/L, where 1 mol = 6.023 x 10^23 of said molecule. Weight per volume is presented as a percent, the percentage being the fraction of the total mass for the specified volume, ie: 300 mL of 20% glucose solution means that 60g of glucose need to be added to maybe 200 mL of water, then bring the total volume to 300 mL.

When performing dilutions, the formula is (c_i)(v_i) = (c_f)(v_f), and solve for the missing number.

Serial dilutions are necessary when diluting to crazy degrees, ie: preparing a 1:100000 dilution of some sample. An ideal way for this example is to perform a 5-step, 10-fold dilution.

When growing bacteria in a flask, ie: Erlenmeyer, grow in LB broth medium with agitation to aerate. The population will grow in 4 phases, and can be charted on a logarithmic graph: lag, log, stationary and death. The bacteria will not be able to take up plasmids during and after the stationary phase. One method of determining which stage the sample is at is to measure the optical density.

ButInTh33nd 04-13-10 08:40 PM

Nucleases are enzymes that cleave phosphodiester bonds between nucleotides. We worked with endonucleases, which cut within the nucleic acids, and are also known as restriction enzymes. The ones we worked with were type II, which do not require ATP and cut at the recognition site. For our course, we only need to know about the ones that recognize palindromic sequences. They can leave three kinds of cuts: 5' overhang, 3' overhang or blunt ends.

They are given names in this form, ie: EcoRI, where E = genus, co = species, R = strain, I = isolated in.

Isoschizmers are restriction enzymes from different sources, but cut the same recognition site.

ButInTh33nd 04-13-10 08:42 PM

The odds of getting a particular sequences of "n" length is 1/4^n

ButInTh33nd 04-13-10 08:46 PM

Gel electrophoresis is a method of analyzing genetic material. The apparatus is a box with agarose gel, and wells at the negative end. The genetic material is injected into the wells, the machine turns on, and because of the overall negative charge of genetic material because of the phosphate backbone of DNA, it will run towards the positive end on the other side based on size and charge. Smaller fragments migrate faster.

ButInTh33nd 04-13-10 08:56 PM

A basic procedure would be to digest the DNA with the restriction enzymes, incubated to 37 C. Loading dye is mixed with the digest, and contains ethidium bromide, a carcinogen that binds to the DNA along the major groove.

Other important details:

Buffer solution must cover agarose gel, but not too much as the current will pass on top of the gel rather than through it. The concentration of the gel also affects migration rates.

Restriction enzyme activity is affected by the buffer composition and the temperature. The buffer needs to be at the correct pH and ionic strength. High temperatures can often be used to destroy restriction enzymes, or add EDTA, which contains Mg2+ ions, that inhibit restriction enzyme activity.

If the digest is performed outside of optimal conditions, you may get star activity, whereby the restriction enzyme ends up cutting at different sites than normal.

ButInTh33nd 04-14-10 03:28 PM

Other types of electrophoresis are DGGE, which measures based on degree of denaturing, and Pulse-field, where the current is run from different directions to increase resolution capabilities.

ButInTh33nd 04-14-10 03:38 PM

DNA Cloning:

Isolate the DNA of interest, combine it with a vector like a plasmid, introduce the recombinant DNA into cells, which replicate, the DNA is isolated and purified.

Plasmid vectors are extranuclear pieces of circular dsDNA, and the one's we use are genetically engineered strains, such as E. coli. Of course lethal potential needs to be neutralized in these strains. Plasmid vectors must feature a sequence cleaned up of non-essential portions, have an origin of replication, multiple restriction sites is useful, and markers used for selection need to be introduced.

A polylinker is a restriction site that can be used by several restriction enzymes.

To assemble the recombinant DNA, digest the plasmid and the DNA segment containing the gene of interest with the restriction enzyme, combine them using the plasmid vector and use DNA ligase to reform the phosphodiester bonds.

ButInTh33nd 04-14-10 03:48 PM

In the experiment we conducted, we treated our strain of E. coli with a solution of CaCl2 to destabilize the cell membrane. We added the plasmid that contained the gene for Ampicillin (an antibiotic similar to resistance. By streaking them in agar plates containing Ampicillin. The colonies that took up the plasmid therefore would be able to grow in the medium, whereas the rest would die.

We used the heat shock method, which is to raise the temperature of the preparation, then plunge it into ice. The molecular mechanism for this is still being studied. The other method we need to know is electroporesis, which uses a very brief electrical current to pass through the medium, essentially punching little holes in the cell membrane to allow the plasmid to pass through.

ButInTh33nd 04-14-10 03:51 PM

Other details:

Antibiotics come in two forms: bacteristats (arrest growth), and bacteriocides (kill cells outright)

The recovery period between when the bacteria have been treated with heat shock, and when they are streaked on the agar plates, is to allow the bacteria to actually express the Ampicillin-resistance gene.

ButInTh33nd 04-14-10 04:15 PM

Two sets of procedures are used to select between bacteria that have no plasmid and bacteria that have the plasmid. A round of screening differentiates between those that have the plasmid, and those that have the recombinant DNA.

Double antibiotic resistance can be used for this purpose.

Another method, a plasmid that has resistance to one antibiotic, and one that has the LacZ' gene and the lac promoter. If the insert is integrated, it interrupts the LacZ' gene and no galactosidase is produced, which turns X-gal blue. Otherwise, They stay white. The white colonies therefore have the insert.

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