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4/15/17
Biotechnology 1015
pBLU Identification
Introduction:
plasmid is a circular piece of DNA in bacteria. It is typically not associated with its circular
chromosome. A plasmid is typically very small, averaging between 3000-5000 base pairs. Most
of the time plasmids do not do anything to the bacteria. However, if they are expressed they may
have genes that are beneficial to the bacteria like antibiotic resistance. A plasmid can be
replicated and horizontally transferred to neighboring bacteria to give them the beneficial genes
as well. Plasmids are important for biotechnology because of their ability to be taken up fairly
easily by bacteria. Scientists can change DNA sequences on plasmids and make them code for
The goal of this experiment was to identify which plasmid was in my unknown sample.
There were three options, pAMP, pKAN, and pBLU. To do this, I looked at different restriction
enzymes cutting sites on all three plasmids. A restriction enzyme is an enzyme that cuts a DNA
fragment at a particular sequence. The sequence is palindromic, meaning it is the same on both
sides of the antiparallel DNA molecule. Each restriction enzyme looks for a particular
palindrome. When a DNA fragment is incubated with an enzyme it has been digested. This is
because the enzyme has digested or cut the DNA fragment. I decided on one enzyme for a
single digest and two different enzymes for a double digest. This will help me identify my
plasmid without a doubt because there will be two different digests to look at and compare.
After the enzymes have digested my plasmid, I will have to run a gel electrophoresis.
This is a type of equipment that separates DNA fragments based on sizes. The smallest DNA
fragments will travel the farthest and the largest DNA fragments will have not travelled very far.
It does this by having an electrical charge. Since DNA is negative, it is attracted to the positive
side of the gel. This is important when looking at DNA because it is too small to see by the naked
eye. It allows you to see the fragment sizes after the restriction enzymes have cut it.
My goal for this experiment is to determine which plasmid I have out of pAMP, pKAN,
or pBLU. This will help me learn laboratory techniques used in a biotechnology lab. Because the
equipment I will be using is used in an actual biotechnology lab. It will also help me learn how to
plasmid, I will perform a single and double digest. I will then run a gel electrophoresis to see my
Methods:
enzymes I want to use for my single and double digest. I needed to pick enzymes that would
allow me to tell the difference between the three different plasmids. I used NEBcutter tool,
http://nc2.neb.com/NEBcutter2/, to determine fragment sizes for each enzyme after each plasmid
had been cut with it. For my single digest I chose Pvul which digest in 10x buffer R. My double
digest used Pstl and Bgll which both digest in 10x buffer O. Theses enzymes were purchased
from Fermentas. The products were Pvul enzyme, Pstl enzyme, and Bgll enzyme. Next, to set up
my digest I needed to determine the concentration of my plasmid. To do this, I loaded 2 ul of the
plasmid onto the nanodrop, after loading a blank. The nanodrop read a concentration of 97.8
ng/ul. I used the formula X concentration*Xul=500ng to determine the amount of plasmid to use.
I determined that I will be using 5 ul of plasmid. Then I set up my digest with the determined
amounts of plasmid, buffer, distilled water, and enzymes to use to reach a total volume of 20ul in
each tube. I decided to use two controls because I will be using two different buffers. This is to
show if the buffer had any effect on the plasmid. My control R had 5 ul of plasmid, 2 ul of 10x
buffer R, and 13 ul of distilled water. My control O had 5 ul of plasmid, 2 ul of 10x buffer O, and
Pstl, 1 ul of Bgll, and 11 ul of distilled water. The total volume in all tubes was 20 ul. After my
digests were set up, they incubated in a 37-degree Celsius water bath for approximately 24 hours.
While my digests incubated, I needed to make a 20x TAE buffer that is essential in
running a gel electrophoresis. To do this I calculated and measured out 19.4 g of Tris base to be
dissolved in 150 mL of distilled water with a stir bar. After the Tris base was dissolved I
measured out 4.6 mL of glacial acetic acid and put it into my solution. I mixed the solution again
with a stir bar. After a few minutes had passed I calculated and measured out 8 mL of 0.5 M
EDTA (pH 8) and put it into my solution. I mixed the solution again with a stir bar. After a few
minutes had passed I brought my solution up to a total volume of 200 mL with distilled water.
Next, I had to determine the pH of my solution to make sure I made it correctly. I calibrated the
pH meter with pH 4, pH 7, and pH 10 solutions. Once the pH meter was calibrated I determined
the pH of my solution, pH 8.46. This is what I had expected it to be. I had to dilute my 20x TAE
to a less concentrated 1x TAE to use in my gel electrophoresis. I used 25 mL of 20x TAE and
diluted it with 475 mL of distilled water to bring to a total volume of 500 mL of 1x TAE.
After my digests have been incubated for approximately 24 hours and my 20x TAE was
diluted to 1x TAE, I was ready to do my gel electrophoresis. I made a 0.8% agarose gel by
microwave for 45 seconds, and an additional 15 seconds. I then added 1 ul of ethidium bromide
to the solution to aid in see my DNA fragments on the gel. Once the bottle that held the solution
was cool to the touch, I poured it into the gel electrophoresis tray with a 6-tooth comb and let it
solidify. Once it was solidified, I pulled out the comb and rearranged the tray so that the DNA
could run to the positive side of the gel. I then loaded in my DNA ladder and digests into the gel.
The DNA ladder was a Fermentas product, titled 1kb DNA ladder. I loaded 6 ul of DNA ladder,
that order). I let me gel run for 35 minutes at 130 V. After my gel had run, I looked at it in the
UV box and took a picture. I noticed some smaller bands were harder to see. I decided to soak
my gel in ethidium bromide for 15 minutes to better see the smaller bands. After the soak, I took
another picture in the UV box. I then used a standard curve and the distance my bands travelled
Results:
My plasmid, code 2834B87, concentration was 97.8 ng/ul. This result allowed me to plan
Digest Setup:
R buffer R
Control 5 ul 2 ul 0 0 0 13 ul 20 ul
O buffer O
Single 5 ul 2 ul 1 ul 0 0 12 ul 20 ul
buffer R
Double 5 ul 2 ul 0 1 ul 1 ul 11 ul 20 ul
buffer O
115
Plasmid: 2834B87 1950, 1750, 700, 500 2100, 1600, 1100
Gel Photos:
Before Soak:
After Soak:
*numbers on right are the fragment sizes (bp) of my DNA ladder.
Standard Curve:
My standard curve ended up having a best fit line that was exponential. Although you
would normally expect a logarithmic, my R2 value showed that it fit an exponential graph better.
As you can see, most data points fall neatly on best fit line.
Conclusion:
After examining all my data, I have concluded that my plasmid, code 2834B87, is pBLU.
My fragment sizes in my single and double digest were too small to have fit any other plasmid
fragments. My single digest clearly had a doublet, to the naked eye, the only possible way to get
a doublet is from pBLU. In my single digest, I had predicted for pBLU a 1980 band and a 1798
(bp) band. Those bands are so close in size they could make a doublet. My double digest was not
as conclusive. There is still room for argument that it could be different plasmid because the
fragment sizes were similar. However, with my double and single digest combined, it is clear to
see that it is pBLU. If I were to do something like this in the future, I would pick enzymes that