Biotechnology 1015
In this essay we will be addressing the following.
1. What is a plasmid
2. How did I address going about figuring out what my plasmid was.
3. What was my randomly selected plasmid is, so here we go.
1.
A plasmid is a small bit of DNA that has been either removed from a cell, or is still
present in non-eukaryotic bacteria. A plasmid can reproduce and replicate separately of that of
the chromosomal DNA and tends to carry vital or non-vital genes needed for survival. Although
however, plasmids are not considered living and by that sense are not subjected to certain
moral issues that come with the alteration of life. Applications of modified plasmids consist of
gene therapy, and counteracting diseases.
A restriction enzymes is an enzyme that takes DNA and cuts it at distinct regions of
nucleotides other wise known as ‘restriction sites’ to do this Restriction endonuclease make
two incisions, both on the phosphate backbone.
By using restriction enzymes, I was able to cut our DNA plasmid at pre-determined regions
(discussed more in Para. _). Most commercially used restriction enzymes first found their way
into laboratory use through the bacteria E. Coli
Gel electrophoresis, is the separation of recombinant DNA Fragments by bp amount and
electro charge. By running a product through a mix of agarose and T.A.E one can watch as
small particles navigate their way through a matrix of desired concentrated gel the smaller the
piece is the faster it moves. By using this and a restriction enzyme you are able to cut up a
piece of DNA and separate the fragments by bp (base pair) size. Gel electrophoresis is used in
almost every laboratory that uses DNA applications, including but not limited to Forensics,
Molecular biology, and genetics.
During this procedure we were presented a first time look at real world applications of
modern day biotechnology. We tested our developed and acquired skills with pipetting and
understanding of lab protocol as well as molecular biology. In this lab we were given on of
three random plasmids; pAMP, pKAN, or pBLU. Using nothing but the assistance of prior
procedures we began testing suitable restriction digests and were told too uncover the
random plasmid were given.
“Here’s DNA tell me what it is.”
The topic of this paper is the importance of correct lab procedures during an exercise
such as plasmid identification. Lab procedures play the utmost critical role in whether or not
an individual can preform a task presented. Improper lab procedures can lead to catastrophic
consequences and complete inaccuracy of results. In this experiment we were testing for the
first time our understanding of pipetting, instrument use, proper dilution, and molecular
concentrations.
So to uncover this, I decided to use several chosen restriction enzymes, digest my
selected DNA, and uncover the remaining fragment sizes. I then compared these to the
expected results of already determined fragment sizes and was awarded my answer.
2.
In this project, we were provided with a random sample plasmid, mine had the code
2110a29. This is the code that provides the instructor with the information on whether or not
my results were accurate by telling him the identity of my DNA
Another important aspect on this lab is concentration amounts, Which were also provided by
the instructor. Although the problem came when we were asked to dilute the stock solution
that was at 500ng/ul to the desired concentration was 150 NG/UL.
After figuring out the required amount plasmid required in a 20ul solution we then added our
restriction enzymes. These products are NEB-Pstl, Ndel, puvl as well as a 3.1buffer
We made three total solutions using the following websites to assist in the procurement and
compatibility; tinyurl.com/plasmidseq, tinyurl.com/NEBcutter, tinyurl.com/nebbuffers,
tinyurl.com/nebdoubledigest, we retrieved the following enzymes1. NDEL+PSTL 2. PVUL 3.
Nothing. We let these digest awhile (60 minutes at 37.5 Celsius) then moved into making a
.08%-.46g/60ml agarose gel with 60ml of 1xTAE (made from a stock solution of 10x TAE with
29.2gtris/2.86ml glacial acidic acid/. 73gEDTA). depending on the thickness of your gel
controls the recommended BP size put into it. Although in our situation .08% was universally
recommended. We then microwaved this and added 1-2ul of ethidium bromide (the DNA
identifier)
Later in our lab during the electrophoresis portion, where we were self tasked into
determining the size of our fragments, we Dyed our enzymes with a total of 4UL of 6x loading
dye, we then used a ladder to compare our fragment sizes with, a ladder is a set of already
determined BP fragment sizes that we then compared with our fragment sizes to the NEB
(New England Biolab) 1k ladder. After adding the ladder to the gel, we then added our DNA in
descending order. After letting it run for 45 minutes at 140v we removed the gel from the
electrophoresis chamber, and using UV lighting to highlight the ethidium bromide attached to
base pairs we identified the Plasmid to be pAMP
Using an exponential line of best fit curve (Microsoft excel) along with a 1k NEB ladder.
3.
The 500NG/UL concentration of DNA has led me into all sorts of riotous fun
Ladder
double
single
control ladder
figure 1.1
FRAGMENT SIZES
figure 1.2
PST1+NDEL
PVUL
pAMP
3134, 1405,
3643, 896
pKAN
2635, 973, 636
3622, 572,
pBLU
2998, 1316, 926, 197
1980, 1798, 726, 480, 453
Figure 1.3
All this and more has lead to me the conclusion that my randomly selected plasmid is pAMP,
because if you look at figure 1.3, the double digest fragment sizes for pAMP barely match with -
-the indicated results on the last two rows of figure 1.2 where if you look at the single digest on
figure 1.2, the last two almost match perfectly with the expected fragment sizes
Credits due too, Professor: Richard Scott
Final conclusion: pAMP.
Cameron Miller
May 04 2014
P.S No Works Cited.