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Chloe Babb

4/15/17

Biotechnology 1015

pBLU Identification

Introduction:

I conducted an experiment to identify a plasmid from three different plasmid options. A

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

something else, like the ability to glow green.

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

identify DNA fragments, which is an important skill used in biotechnology. To identify my

plasmid, I will perform a single and double digest. I will then run a gel electrophoresis to see my

DNA fragments and determine cutting sites.

Methods:

My first step in identifying my plasmid, code name 2834B87, is to determine which

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

13 ul of distilled water. My single digest had 5 ul of plasmid, 2 ul of 10x buffer R, 1 ul of Pvul,

and 12 ul of distilled water. My double digest had 5 ul of plasmid, 2 ul of 10x buffer O, 1 ul of

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

measuring out 0.3256 g of agarose and dissolved it in 40 mL of 1x TAE by heating it up in my

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,

20 ul of control R, 20 ul of control O, 20 ul of my single digest, and 20 ul of my double digest (in

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

to identify my fragment sizes.

Results:

My plasmid, code 2834B87, concentration was 97.8 ng/ul. This result allowed me to plan

my digests using calculations to determine how much plasmid to use.

Digest Setup:

Plasmid 10x Pvul Pstl Bgll dH2O Total


Buffer Volume
Control 5 ul 2 ul 0 0 0 13 ul 20 ul

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

I determined my enzymes by making predictions on fragment sizes depending on their

cutting sites using NEBcutter tool, http://nc2.neb.com/NEBcutter2/. I then ran my digests on a

gel to determine my fragment sizes.

Enzyme Predicts and Gel Results:

Single Digest: Pvul fragment Double Digest: Pstl and Bgll

sizes (bp) fragment sizes (bp)


pAMP 3643, 896 2078, 1185, 1118, 158
pKAN 3622, 572 1903, 923, 794, 313, 261
pBLU 1980, 1798, 726, 480, 453 2121, 1615, 1191, 197, 188,

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

made large are more distinct bands.