Monday, February 2, 2009

Lectures - DNA repair + Control of gene expression

Between last monday and today we have finished the section on DNA repair and have covered most of the section on control of gene expression.

Last Monday (Jan 26) we closed the DNA repair section, covering the concept of emergency DNA repair: When extensive damage in the DNA is detected by RNA polymerase and is repaired not just by the regular DNA polymerase, but also by a battery of DNA polymerases that are less accurate, but more specific for a type of damage. They also lack proofrweading capacity, so the likelihood that there will be mistakes during the repair process is higher than with other DNA repair mechanisms.

Between Friday (Jan 30) and today, we have been studying the basics of control of gene expression. So far we have focused on transcriptional control, and on Monday we will focus on mechanisms of post-transcriptional control.

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Lab 12 - Small-scale plasmid DNA purification (minipreps)

Thursday Jan 29 2009

We went back to the bacterial cultures we had of transformed bacteria (E. coli), to reverse (in a way) the process we started. In this case we want to isolate the plasmid (Bio-Rad's pGLO) that we used to genetically transform the bacteria. In the process of cloning DNA this is one of the steps you follow to study the DNA segement of interest, in our case the GFP gene contained in the pGLO plasmid. We made zillions of copies of it, and now we have to extract it from the bacteria to analyze it.

We used Promega's Wizard® Plus SV minipreps DNA purification system. An easy to use kit to purify plasmid DNA in a lab like the one we have available.

Next week we'll run a confirmation gel and perfomr a restriction enzyme digestion (RED) of the pGLO plasmid.
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Lab 11 - Polyacrylamide Gel Electrophoresis (PAGE) of GFP samples

Thursday Jan 29 2009

This lab should have taken place on Wednesday Jan 28, but classes were cancelled due to weather.

Polyacrylamide Gel Electrophoresis (PAGE) is a technique used for separating polynucleotides or polypeptides that are very similar in size, providing greater resolution than with an agarose gel.

We used PAGE to measure the size of a protein, GFP. If we follow the series of "make believe" in which we are dealing with a new protein, this would be a step to find more information about it. Protein size is quantified in Daltons (Da), a measure of molecular mass. One Dalton is defined as the mass of a hydrogen atom, which is 1.66 x 10-24grams (g).

PAGE is used for separating proteins ranging in size from 5 to 2,000 kDa due to the uniform pore size provided by the polyacrylamide gel. Agarose gels can also be used to separate proteins, but they do not have a uniform pore size, so they are optimal only for electrophoresis of proteins that are larger than 200 kDa. We ran two types of PAGEs: Native and denaturing (a.k.a. SDS)

Proteins can have varying charges and complex shapes, therefore they may not migrate into the gel at similar rates, or at all. In native gel electrophoresis the proteins being separated differ in molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Because of different charges and tertiary structure proteins of the same mass may migrate at different rates.

In SDS gel electrophoresis proteins are denatured (linearized) in the presence of a detergent such as Sodium Dodecyl Sulfate (SDS) that coats the proteins with a negative charge. The resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which they migrate in the gel is relative only to its size (molecular weight) and not its charge or shape.

We will be able to compare teh results in both gels, and if GFP has any activity in either one of them (through pictures taken under UV light).
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