1. What’s the difference between transformation and transfection of a cell? (4 points)
Answer: Transformation is done on the cells to introduce a new DNA. In most cases, transformation is done on the bacterial cells by insertion of a target DNA containing plasmid, whereas, transfection is almost exclusively done on the mammalian cells to express protein from that target DNA containing plasmid. Here, transfection word came from the blending of two words, trans and infection. Therefore, in brief, a eukaryotic cell is infected by a foreign plasmid in the transfection method.
2. Give 2 reasons why one would transform a cell with a plasmid? Give 2 reasons why one would transfect cells. (8 points)
Answer:
Reasons for the transformation:
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In the plasmid DNA purification protocol, which step(s) remove(s) chromosomal DNA? How are the ingredients in the L7 Lysis Buffer accomplishing the goal of this buffer? Mechanistically, what is the spin column doing? (10 points)
Answer: Chromosomal DNA became renatured and entangled with cellular debris after addition of precipitation buffer (N4), whereas, centrifugation step separated the chromosomal DNA from the plasmid containing supernatant.
L7 lysis buffer has a base (NaOH) and a detergent (SDS) in it. Sodium dodecyl sulfate (SDS) is an anionic detergent, which ruptures the cell membrane, whereas, NaOH helps to break down the cell membrane and denature the DNA of the cells.
The spin column has a silica membrane in it, which can bind tightly with charged backbone of the plasmid DNA, whereas, washing that silica membrane with a salt solution will release the plasmid DNA from the spin column.
8. For the “calcium phosphate” transfection method, which type of Ca 2+/PO4 3-/DNA precipitate is best: Large flakes or small? Why? (6
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Reference: Cornil, C. A., & Ball, G. F. (2008). Interplay Among Catecholamine Systems: Dopamine Binds to a2-adrenergic Receptors in Birds and Mammals. The Journal of Comparative Neurology, 511(5), 610–627.
11. A pharmacogeneticist excitedly discusses with you her discovery of a mu opioid receptor (muOR) single nucleotide polymorphism (SNP) and its potential physiological relevance. The SNP mutation encodes replacement of a phenylalanine residue at Position 293 in the muOR protein with an alanine side chain. Individuals carrying this SNP are 10 times more likely than normal (wildtype muOR) individuals to die following muOR agonist (heroin or oxycontin) overdose. You study muOR pharmacology and mention to your colleague that you would like to test the hypothesis that this Phe293Ala SNP could be affecting one or more ligand binding sites of the muOR. As a first step, you create the Phe293Ala muOR mutant cDNA and subclone it into a plasmid cDNA that also encodes ampicillin
While the previous experiment identified colonies containing recombinant DNA, the patching experiment distinguished which colonies contained the hlyA fragment and which ones did not. Colonies that could cause haemolysis of the blood agar plate indicated that recombinant DNA taken up contained the hlyA fragment ligated with pBluescript, which is the desired subcloning product. The hlyA fragment contains the hlyA gene which encodes for a haemolytic protein that causes the red blood cells in the blood agar to lyse. Therefore, non-haemolytic colonies were transformed with pBluescript plasmid ligated with the pK184 fragment and were not able to cause haemolysis as no hlyA gene was present. In theory, this experiment allowed for the aim to be achieved as it identified colonies with the desired product. Inoculating certain colonies in broth culture allowed for gel electrophoresis to be carried out and confirm if the aim of the experiment has been
The varieties of pharmaceutical and prescription drugs that are available to the public provide many different consequences, which could lead to other health problems among users. Opioids, for example, are typical...
Therefore colonies containing the non-recombinant pUC19 plasmid have a functional lacz’ gene appear blue on the agar and colonies containing recombinant pUC19 would have a non-functional lacz’ gene due to insertional inactivation and appear white on the growing medium.
the strands to seperate, then cooled to allow the primers in the solution to b...
These six samples (crude -/+, broken -/+, and whole -/+) were spun at 5000 rpm, and the resulting pellets were isolated and resuspended in DNase buffer. The set of suspensions labeled with a (+) was incubated in DNase enzyme for 15 minutes, and afterwards incubated in 15 uL of STOP solution. All six samples were lysed for DNA extraction with DNA extraction buffer, and micro-centrifuged at maximum speed. To precipitate the extracted DNA, the supernatants from each of the six samples were added to their correspondingly labeled micro-centrifuge tubes containing 7% ethanol (Parent et. al, 2008To bind the DNA, the ethanol lysate mixtures were transferred to labeled spin columns and spun for one minute in the micro-centrifuge at maximum speed. To wash the bound DNA, the spin columns were washed and spun three times at maximum speed. In order to elute the bound DNA, the samples were washed in 80 uL of distilled water and spun again for 2 minutes at maximum speed (Parent et. al,
Almost one hundred years ago, prescription drugs like morphine were available at almost any general store. Women carried bottles of very addictive potent opiate based pain killers in their purse. Many individuals like Edgar Allen Poe died from such addictions. Since that time through various federal, state and local laws, drugs like morphine are now prescription drugs; however, this has not stopped the addiction to opiate based pain killers. Today’s society combats an ever increasing number of very deadly addictive drugs from designer drugs to narcotics to the less potent but equally destructive alcohol and marijuana. With all of these new and old drugs going in and out of vogue with addicts, it appears that the increase of misuse and abuse is founded greater in the prescription opiate based painkillers.
Volkows, N. D., & Muenke, M. (2012). Human Genetics. The genetics of addiction, Vol 131(6), 773-777. Retrieved from http://dx.doi.org/10.1007/s00439-012-1173-3
Opioid’s chemical composition consist of many highly addictive substances which cause the human body to become quickly tolerant. Many opioid users become addictive to the substance because the doctors have been over prescribing. “In the United States, there were 14,800 annual prescribed opioid (PO) deaths in 2008” with the US having less restrictions (Fischer, Benedikt, et al 178). The United States have implemented more regulations so that “high levels of PO-related harms been associated with highly potent oxycodone formulas” will decrease (Fischer, Benedikt, et al 178). With the regulations, it does not change the fact that opioids are is destructive. The regulations assistance by lessening the probability of patients becoming addictive to opioid. There are numerous generations that are effected and harmed by the detrimental effects of opioids on opioid-dependent patients.
Either transduction or transfection can be used to get the therapeutic genes into the patients system. Transfection is when the genes are introduced physically or chemically in a way that allows the cell membrane to be temporarily permeable to a foreign DNA. In the second method used for gene therapy, transduction, there is a beneficial gene added into the genetic material of the virus, which then is allowed to infect the target cell which is the indirect transfer method for gene therapy.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
The ultimate goal of pharmacogenomics, as stated by Henig, “would be for everyone’s genome to be analyzed indi...
Gene therapy, a relatively new innovention, is becoming popular across the country. Gene therapy modifies a part of an organism, whereas cloning creates an entirely recreated organism. This technique can be conducted in vivo in either somatic or germ cells. The process is essentially aimed at fixing a genetic disorder or disease by inserting a functional gene to replace the faulty one (Houdebine 2003). Many methods to conduct a gene transfer have been tested. The two types are in vivo and in vitro. Transferring genes in vivo means placing the functional genes directly into the target tissue; while vitro transfers creates the genes outside of the body, in Petri dishes. Vitro is an expensive process that r...
Also, involved in chemistry are dopamine and norepinephrine, chemical cousins of amphetamines. Dopamine, a neurochemical released by PEA, makes us feel good.(1) A recent study done at Emory University shows that female voles (small rodents) choose their mates in response to dopamine being released in their brains. When injected with dopamine in a male vole's presence, the female will pick him out of a crowd later.
By the year 2000 opioid medicine containing oxycodone etc., are being abused and misused and more than doubled in 10 years’ time.
Mammalian dopamine auto receptors are G-protein coupled receptors (GPCRs) on membranes of neurons. Dopamine receptors are broken into two different groups, D-1 like (D1 and D5) and D2-like (D2, D3, D4). D2-like receptors are found primarily in the midbrain and mediate inhibitory neurotransmission. Increased levels of D2 receptors have been associated with disorders, including both Parkinson’s and ADHD (Anzalone,2012).