A. This is what I would expect running a plasmid purified by a isolation prep similar to what was discussed in the lab.  The plasmid purified from the cell lysate will occur in a variety of supercoiled forms.  Despite the identical molecular weight of the DNA in the different supercoiled forms, more supercoiled forms will occupy a smaller amount of volume and will run faster on an agarose gel. This result does not indicate the presence of any contaminants or a procedural mistake.

Source: http://faculty.plattsburgh.edu/donald.slish/Electrophoresis.html

B. The size of the plasmid run in this example cannot be reliably determined from the picture of the gel that has been provided.  This is due to the various shapes of the DNA which can be linear, supercoiled, or circular.  A plasmid in a highly supercoiled state will occupy a smaller amount of space than that same plasmid in a linear or circular conformation, which will cause it to run faster on a gel than its true molecular weight.  To determine the correct size (kb) of the plasmid the sample would have to be treated with a restriction enzyme to create a linear DNA strand, or the gel would have to be run under denaturing conditions.

Source: http://faculty.plattsburgh.edu/donald.slish/Electrophoresis.html
Source 2: http://en.wikipedia.org/wiki/Agarose_gel_electrophoresis

C. The location of the hypothetical C band indicates the presence of a very large contaminant.  Taking into account the protocol used in the isolation of this DNA it is most likely that this contaminant would be chromosomal DNA.  It is important when isolating plasmid DNA to treat the sample carefully at all steps before the physical removal of the chromosomal DNA with the other large cellular debris.  Likely this sample was too vigorously agitated immediately after lysis, allowing some of the chromosomal DNA to remain in solution instead of being removed by centrifugation.

D. The location of this D band indicates the presence of a small and irregularly sized contaminant that has run very quickly through the gel.  This is most likely partially digested chromosomal DNA if the DNase has not been properly removed, but could also be RNA that has come down with the plasmid.  DNases should be inactivated by SDS and EDTA, so there may have been a mistake in the creation of solutions A and/or B.  RNA would be digested by RNase in the very last step of the protocol, but if  it was not added RNA could stay in the sample.

A. The resuspension of the culture was performed in a SET solution comprised of 25% sucrose, EDTA, and Tris in the presence of lysozyme.  This treatment eats at the thick peptidoglycan layer surrounding many gram-positive bacteria.  The EDTA in the SET solution chelates ions from the lipid membrane of the cell, weakening it, while the sucrose solution serves to prevent osmotic forces from prematurely lysing the cell.  This treatment leaves the weakened protoplast, which can be readily lysed in conditions where DNase activity can be controlled.  After neutralization the DNA is partitioned by addition of phenol-chloroform-alcohol solution and again with only a chloroform-alcohol solution.  These steps are followed by alcohol precipitation of the DNA.

Source: http://en.wikipedia.org/wiki/Protoplast


B. Advances were found using this protocol that allow for: reduction of plasmid isolation times from 6+ hours down to 1-3; purification of plasmid DNA from sporulated cells; ability to replicate the plasmid without having to treat with restriction enzymes; reduction of the amount of contamination by chromosomal DNA; ability to transform other cells (unreliable earlier due to amount of contamination in samples).  These procedures gave researches a way to isolate both high- and low-copy plasmids from gram positive cells.


C. Chloramphenicol is a bacterio-static antibiotic, which operates by interfering with a subunit of the ribosome and thus preventing the translation of RNA into protein.  It does not interfere with replication of DNA, though in some papers it is presented that it induces preferential replication of small plasmid DNA's over chromosomal DNA, likely due to saturation of the replication enzymes by plasmid templates.  This inhibition of protein synthesis and cell growth, while DNA replication continues, increases the number of plasmids present within the cell.

Source: http://www.bio.net/bionet/mm/methods/1997-May/057280.html
Source 2: http://www1.qiagen.com/FAQ/faqview.aspx?faqid=3&SearchText=chloramphenicol&FaqCategoryId=0&MenuItemId=0&ProductLineId=0
 

A. Lectin chromatography is used to purify priteins which contain sugar groups that have been added post-transcriptionally.  Lectins bind to specific sugars contained in glycoproteins and not to proteins that have no attached sugars.  Lectin affinity chromatography is well suited for purifying glycoproteins and would be a very poor choice for non-glycoproteins.


B. Amylose based purification requires the attachment of a Maltose Binding Protein to the N terminus of the protein to be purified.  The MBP portion of the protein will bind with high affinity to the amylase in the matrix of the column.  The amylase-bound protein can be displaced from the resin by a solution containing maltose.  This system can be used to purify recombinant proteins with MBP attached.

Source: Expression and Purification of Recombinant Proteins by Fusion to Maltose-Binding Protein; Paul Riggs; Molecular Biotechnology; Vol 15; 51-63
    

C. Avidin based protein purification systems rely on the highly specific binding of avidin to biotin.  The protein to be purified is attached with a recombinant avidin, and passed through the column where it binds to the immobilized biotin.

Source: The avidin-biotin complex in affinity cytochemistry; Edward A. Bayer; Methods in Enzymology vol 62; 308-315


D. Heparin based columns work in two main ways.  The heparin is a polysaccharide that binds strongly with a wide range of proteins, as well as a strongly negatively charged molecule that work as a cation exchange system that can bind positively charged proteins.  Heparin fractionation is best suited for isolating either polysaccharide binding proteins or proteins with strong positive charges which will interact with the heparin charge distribution and remain in the column after other contaminants have been washed out.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-064-9_18
 

Due to the nature of metal binding proteins it would be best to avoid using a nickel column with a his-tagged protein.  The protein may bind too strongly to the exposed Ni of the resin and require extreme elution solutions.  It would also be poor choice to use a cation-exchange fractionation because the exposed charges in the resin could actually repel the metal binding proteins, anion-exchange would be a better choice.

I have no particular source here, just reasoning out the affinity of metal binding proteins for metals and the fact that metals are positively charged elements which would require negative charges in the active site that would be repelled the negative charges on a cation exchange resin.
 

A. Human growth hormone is 191 amino acid long protein produced in the human body that boosts activity of many growth inducing systems including bone, muscle, and organ growth; and its deficiency can produce many symptoms that are associated with reduced health and a lower ability of the body to undergo anabolic pathways leading to reduced growth.  Being able to recombinantly produce this protein allows for the treatment of a low growth hormone level with exogenous HGH, which can largely reverse the effects of low growth hormone.

Source: http://en.wikipedia.org/wiki/Human_growth_hormone


B. HGH is produced in the human body in the anterior pituitary gland following stimulation by growth hormone releasing hormone.

Source: http://en.wikipedia.org/wiki/Growth_hormone_releasing_hormone


C. The major difficulty in using HGH from human sources is the very low production of it in the body.  The reference range for serum HGH (range consituting +/- 2 standard deviations) is 0.01-10.00 ng/mL for healthy adult humans depending on sex.  Clearly it would take a very large amount of blood plasma to be able to purify a useful amount of HGH from human sources.


D. The recombinant host for bioidentical growth hormone is E. coli.

Source: http://en.wikipedia.org/wiki/Humatrope


E. There has been controversy about an FDA decision to allow the presciption of recombinant growth hormone to children who are within normal ranges of height for their sex and age.  This presents both safety and ethical concerns regarding this drug.  Prescription to treat children who are not below normal levels of height could have negative health effects years later, while it encourages doctors to perpetuate the myth that taller is better.

Source: http://www.foxnews.com/story/0,2933,99277,00.html
 

A. I think formamide protocols apply to eukaryotic organisms as prokaryotic organisms don't typically have large enough genomes to be well affected by the formamide based DNA prep protocol that I have found.

Source: http://en.wikipedia.org/wiki/Formamide

B. Formamide reduces the Tm of large DNA molecules, which allows the conduction of isolation of DNA at lower temperatures that is required without formamide present.  This reduces the activity of Dnases, allowing for better purification of undegraded DNA.  Formamide allows the purification of large DNA strands  This allows the researcher to lyse the cells, denature the DNA in presence of formamide to stabilize large DNA and prevent irreversible denaturation and dialize out small contaminants.

The protocol uses formamide to stabilize DNA while allowing proteinase K to digest the proteins in the lysate then dialyzing the sample in colloidal bags which allows most cellular debris to filter out while retaining the chromosomal DNA which is too large to pass through the bag.

Source: Nucleic acid reassociation in formamide; Betty L. McConaughy; Biochemistry, 1969, 3289-3295
Source 2: Isolation of High-Molecular-Weight DNA from Eukaryotic Cells
by Formamide Treatment and Dialysis - ANALYTICAL BIOCHEMISTRY 164,53-59 (1987)