Ally Mortensen

Mr. Tuttle

Biotech 1015

05/23/2016

Plasmid Identification Report

Plasmid Code 6892C22

For this experiment, the general topic was Plasmids. A plasmid is a genetic structure

within a cell that can independently replicate without chromosomes, and are commonly used in

the process of manipulating genes. Just as well, restriction enzymes were also an important part

of this experiment. Restriction enzymes are used as cutters, in which they are placed into DNA

and will cut the DNA at a specific area, known as a cut site. The restriction enzymes allow for

new DNA or genes to be placed within DNA, which is usually known as genetic engineering.

Also for this experiment, gel electrophoresis was used, which is an agarose gel that is placed in

an electrophoresis box, with the wells facing the negative pole of the box. The wells are filled

with the DNA solution that is being tested, and then the gel is ran. When the gel runs, the DNA

solutions begin to spread out, depending on the sizes of the molecules within the DNA, and will

create very specific and unique placement of bands. Overall, the scientific goal of this

experiment was to use our skills that weve acquired over this school year, and put them to use in

order to properly identify a mystery plasmid that we were given. The general goal of this lab is

important to my understanding of biotechnology lab techniques, so that I at least know the basis

of what needs to be done in order to complete the task given. The strategy that I used for this

experiment, was to create an agarose gel and to run a basic restriction digest, while
simultaneously doing the same experiment virtually. After receiving my results, I would compare

them and identify the plasmid.

The code name of the plasmid I received was 6892C22. I determined the concentration of

my plasmid by micro-pipette mixing the solution, and then determining how much liquid was in

the tube using a micropipette. My results were that the concentration of my plasmid was 3.0ug

per 34 microliters. The source of the marker that I used was the Lambda with HindIII digest,

manufactured by BIO-RAD. I used the rectriction enzymes EcoRI, made by BIO-RAD, PstI,

made by BIO-RAD, and HindIII, made by BIO-RAD. When running these digests my marker

tube contained 8L of Lambda with HindIII and 2L of loading dye. My single digest tube

contained 5L of restriction buffer, 1L of HindIII, 2L of loading dye, and 4L of my plasmid.

Finally, my double digest tube contained 5L of restriction buffer, 1L of EcoRI, 1L of Pst1,

2L of my plasmid, and 2L of loading dye. I placed all three of these tubes in the water bath for

30 minutes at 37 degrees Celsius. I made my agarose gel using a 1.4%, and made it using 1xTAE

buffer, .7g agarose, and D-water. I ran my gel for 45 minutes at 100 volts. The buffer I used for

running my gel was a 1x solution that was obtained from my 20x stock that I created at the

beginning of the lab, which had 19.48g of Tris base, 150 mL of D-water, 4.6mL glacial acetic

acid, and 10mL EDTA. After the experiment was complete, I recorded my images of the

experiment using my iPhone 6s Plus. I determined the fragment sizes of the DNA fragments in

my gel by running a virtual gel online, in order to get accurate results. I predicted the fragments

would appear very differently from each other within each digest, using the NEB cutter and a

plasmid sequence reference online.

When the experiment began, the concentration was 3.0ug per 34 microliters. Below are

all of my results from the experiment.

 Figure 1: My results for my gel, the
lanes from left to right being my
Marker, my Single Digest, and my
Double Digest.

Figure 2: My results from my online Figure 3: My results from my online

digest, what the gel should look like digest, what the gel should look like
with a single digest of HindIII with a double digest using PstI and
EcoRI

ONLINE DIGEST RESULTS

pAMP pKAN pBLU

EcoRI 2137 EcoRI 2116 EcoRI 3139
PstI 946 PstI N/A PstI N/A
HindIII 233 HindIII 234 HindIII 3190
Plasmid Size: 4539 Plasmid Size: 4194 Plasmid Size: 5437
I was unable to clearly identify the actual fragment sizes of the plasmids using my physical gel,
but the table above demonstrates the fragment sizes for the digests ran online.
After comparing my results, I have come to the conclusion that the plasmid I was given is the
pBLU plasmid. I compared the results from my physical gel to that of the gels online, and the
two that are very similar in band length would be the pBLU plasmid.
 References
Plasmid definition
https://www.google.com/search?sourceid=chrome-psyapi2&ion=1&espv=2&ie=UTF-
8&q=what%20is%20a%20plasmid&oq=what%20is%20a
%20plasmid&aqs=chrome.0.0l6.1947j0j7

Restriction Enzyme Definition

https://www.google.com/search?
espv=2&q=what+is+a+restriction+enzyme&oq=what+is+a+re&gs_l=serp.1.0.0i67j0i20j
0i67j0i20j0l6.73168.73450.0.74995.3.3.0.0.0.0.156.262.0j2.2.0....0...1c.1.64.serp..1.2.26
2.p3w3Bk8P96M

Plasmid Sequences
Cold Spring Harbor Laboratory
https://www.dnalc.org/resources/plasmids.html

NEB Online Cutter

New England BioLabs
http://nc2.neb.com/NEBcutter2/