Research Proposal
1. Why research Staphylococcus
Aureus DNA extraction?
In general, experimental
research is important because it allows us to reinforce what we already know
about organisms, but it can also open the door to new discoveries. For example,
defining the best technique to extract DNA material from an organism can either
confirm a hypothesis made on existing knowledge or it can lead us to the
discovery of new information. In addition, determining the best method to
extract DNA from a specific organism can be found valuable to an institution,
as it can serve as a basis for the development of further investigations made
on that subject. I will be conducting several experiments to extract DNA material
from the Staphylococcus Aureus bacteria cells. Staphylococcus Aureus or S. aureus for short, is a bacterium
that can be part of the normal skin flora of human beings and never cause a
problem; it is estimated that 33% of Americans carry this microbe without
illness (MRSA Tracking). However, if given the opportunity S. aureus will attack the body and cause illness; and what’s worse,
is that many strains have developed resistance to widely used antibiotics such
as Vancomycin and Methicillin making this seemingly harmless organism deadly
under certain conditions (Gram-positive bacteria). Analyzing the DNA of this
bacterium to understand how it developed this drug resistance is another
important reason to figure out what is the best way extract its DNA as best as
possible.
2. Research tactics.
There will be a total of 6 DNA
extraction protocols (Determining an), one of which will be a commercial kit
guaranteed to render high yields of quality DNA material (Ready-LyseTM lysozyme).
Then I will analyze the extracted material using a microvolume UV-Vis
spectrophotometer to quantify the results. The results that I will be observing
are the concentration of DNA material and also the quality of the material
extracted (260/280 and).
3. Variable table
Name
|
I/D/C
|
Symbol
|
Units
|
Description
|
Nucleic Acid
Concentration
|
D
|
ng/uL
|
This figure will
depend on the protocol used to extract DNA. The higher the value the more DNA
material there was extracted.
|
|
Nucleic Acid Purity
|
D
|
260/230
|
unit-less ratio
|
This figure will
help determine the ratio of pure DNA material there is through the extraction
in contrast to organic contaminants such as proteins. The ideal 260/280 value
is ~1.8, arbitrarily.
|
Method of Extraction
|
I
|
Protocol no.
|
There will six different
methods used to extract the bacterium’s DNA and will identified as
“Protocol…” followed by number 1, 2, 3, 4, or 5; for practical reasons.
|
|
Bacteria Sample
|
C
|
The bacteria used to
determine the effectiveness of each extraction method will all come from the
same isolated colonies (petri dish).
|
||
Equipment
|
C
|
Equipment used for
centrifuging, heating, cooling, and measuring will all be the same throughout
the different tests and trials thereafter.
|
||
Chemicals
|
C
|
Chemicals, such as
alcohols and buffers will all come from the same source.
|
4. Overview of findings
Research question-
When comparing the quantity and quality of
the DNA material extracted with the following methods: Boiling Protocol, TE
Boiling Protocol, TE Freeze and Thaw Protocol, Lysis Protocol, and Meat
Tenderizer Protocol, which DNA extraction protocol worked better? Which one
extracted higher yields? Which one extracted better quality DNA?
Hypothesis-
I think that Meat Tenderizer Protocol will extract
better quality DNA in higher yields.
References
Developing an effective E-coli
protocol. (n.d.). Raw data in preparation.
Gram-positive bacteria. (2011). In K. Rogers (Ed.), Bacteria and viruses. Retrieved from
Gale Virtual Reference Library database. (Accession No. GALE|CX4040900042)
MRSA tracking. (2016, march 3). In Centers for disease control and prevention. Retrieved December 16,
2016, from https://www.cdc.gov/mrsa/tracking/
Ready-LyseTM lysozyme solution [Pamphlet]. (2012). Retrieved from
http://www.epibio.com/docs/default-source/protocols/ready-lyse-lysozyme-solution.pdf?sfvrsn=6
260/280 and 260/230 ratios [Pamphlet]. (2008). Retrieved from
http://www.nanodrop.com/Library/T009-NanoDrop%201000-&-NanoDrop%208000-Nucleic-Acid-Purity-Ratios.pdf
