Plasmid Identification Project

Calvin Dam
Period 5

What are plasmids and what are plasmids used for? To understand the
experiment that Im performing and why I am performing the experiment I must explain
several terms. Plasmids are a small, circular, double-stranded dna molecule. When a
bacteria cell divides, all of the plasmids in the cell are copied so both offspring cell
receives a copy of the plasmid. This enables scientists to use plasmids to clone,
transfer, and manipulate genes. Researchers can place dna fragments or genes into a
plasmid. Then, because bacteria replicates quickly, they can be used as factories to
copy dna fragments in large amounts. In this experiment I am given unknown plasmid
and must calculate what it is by cutting the plasmids with Restriction Enzymes and
performing Gel Electrophoresis.
Restriction enzymes are DNA-cutting enzymes found in bacteria cells and cut
dna into smaller fragments. However restriction enzymes only cut at specific sequences
in the dna. This results in sticky ends and allows other molecules with similar sticky
ends to join together and create recombinant DNA. In my experiment I must find out
where the enzymes have cut by using gel electrophoresis and compare my data to with
the expected places the enzymes are suppose to cut.
Gel Electrophoresis is a process where we separate Dna based on base pairs.
By placing dna in agarose gel and then running electricity through the gel. Since dna
has a negative overall charge by running electricity through a solution the dna shall
move towards the positively charged side of the Gel Box. Since dna with less base pairs
move faster than dna with more base pairs we are able to determine where the
restriction enzymes cut by finding out how long each dna fragment is.
The overall goal for this experiment is by using restriction enzymes to cut
plasmids and by using gel electrophoresis to see where the enzymes have cut to solve
what is my unknown plasmid that I have been given out of pAmp, pKan, and pBlu.
Achieving this goal is useful due to it proving that I am able to perform digests with
restriction enzymes on plasmids and that Im able to where each enzyme has cut. Im
able to achieve my goal by mixing the plasmid, buffer, and enzymes together in one
solution then running gel electrophoresis on the solution. From the gels results I can
predict which plasmid I have received for the experiment.

Kind of like cooking to reach my end goal I have used different ingredients and
followed several steps to achieve my end results. My plasmid that Ive been given code
name is 7430A69 and the concentration of the plasmid is 150 ng/ microliters which was
given to us. My marker (ladder) dna was a 1k Ladder and was from NEB. Also the
restriction enzymes I obtained are named BGlI, Pstl, and Pvul. They were also
produced by NEB. While the buffer I used was Buffer 3 and manufactured by NEB. For
my digestion I had 2 different digestion reactions; a single and a double digest. My
single digest consisted of the BglI enzyme while my double digest had the PstI and the
PvuI enzymes. I incubated the enzymes for about 1 hour at 37 degrees Celsius. While
the enzymes were incubating I created a 0.8% agarose gel by adding 0.48 grams of
solid agarose and mixed it with 60mL 1X TAE that has been diluted from 10X TAE. I
then microwaved the mixture for 45 seconds plus two 15 seconds increments. (45
sec+15 sec+ 15 sec). After heating the gel I then added 5 microliters of ethidium
bromide. After placing the gel and letting it solidify I then poured 280 mL of 1X TAE
(Diluted from 28 mL of 10X TAE) as the buffer over the gel. I then ran the gel for 40
minutes at 140 voltage. After running the gel I then placed it in the UV imaging system
and took a picture of it. I was able to find out the sizes of the Dna fragments by
comparing the ladder bands and the digests bands in a standard curve graph. I was
also able to predict where the fragments were cut by using various programs:
tinyurl.com/plasmidseq for plasmid sequences, and tinyurl.com/NEBcutter. for my
single and my double digest cuts.

Now to recap my Plasmids codename is 7430A69 and the concentration is 150
ng/ microliters.


Legend

Lanes: (With bands)
Lane 1: Ladders
Lane 2: Control
Lane 3: Single Digest
Lane 4: Double Digest
Lane 5: Ladder

Ladder Sizes
1st ladder: 10,000 Base Pairs (bp)
2nd Ladder: 8,000 bp
3rd Ladder band: 6,000 bp
4th Ladder band: 5,000 bp
5th Ladder band: 4,000 bp
6th Ladder band: 3,000 bp
7th Ladder band: 2,000 bp
8th Ladder band: 1,500 bp
9th Ladder band: 1,000 bp
10th Ladder band: 500 bp





The line fits the data well because the R^2 is 0.9977 and on the graph the line goes
through almost all of the data points center.


To answer this final project I assume that the plasmid I have experimented on is the
pBlu plasmid. My reason being is that the DNA fragment sizes for my experimental
plasmid with a single digest, specifically the 2356 and 1772 (bp) fragments are the
closest to the pBlu (Expected) sizes which are 2121 and 1740 (bp). This also holds true
to my double digest where the fragment sizes 1876 and 1674 are closest to the pBlu
which are 1980, and 1316 (bp). So I conclude that I was given the pBLU plasmid.