Cover page for BTEC1015 Plasmid Identification Report
Both a printed and an electronic version of your report must be submitted to
your instructor.
In order to prevent possible unintentional plagiarism, this pledge must be attached
to the front of your Plasmid ID report. The consequence of plagiarizing is a zero on
the assignment and a possible (even probable) failing grade in the course. Please
refer to the links below to acquire more information on how to avoid plagiarism.
http://iechs.org/docs/AcademicHonestyPolicy.pdf
http://owl.english.purdue.edu/owl/resource/930/01/
Students’ pledge:
This report is entirely my own written work. I have checked each sentence of my
report (especially the introduction) and attest I have not copied and pasted
sentences or phrases from any source, including the Internet or another student. I
also understand that Wikipedia, while possibly useful for gaining a preliminary
understanding of a topic, is not a citable source. I understand that I may use
information given to me by my instructor, but that this should be written in my own
words and probably requires a citation. I have used the information in the Plasmid
Report Guidelines to properly cite my sources.
NAME: DAVID JOHN BARLOW
SIGNATURE:DAVID
BARLOW 12/09/2011
David Barlow
Biotechnology 1015
11/25/2011
Biotechnology 1015 Plasmid Identification Project
IntroductionA plasmid, “is a small, Circular self-replicating double-stranded DNA molecules
found primarily in bacterial cells”(Thieman 2009; Palladino 2009). Restriction enzymes,
“are used to cut DNA or plasma in a specific sequence (restriction sites) and are mainly
found in bacteria ”(Barlow 2011, pers. comm.). Gel electrophoresis, “ is a laboratory
procedure that involves using an electrical charge to move and separate biomolecules of
different sizes, such as DNA, RNA, and proteins through a semisolid separating gel
matrix. Examples include agarose gel electrophoresis and polyacrylamide gel
electrophoresis (PAGE) ”(Thieman 2009; Palladino). The goal of this experiment is to
identify a unknown plasmid out of four plasmids (pAMP,pBLU,pGEM,pGLO). This
experiment has one main purpose to Salt Lake Community College, there purpose is to
evaluate if a student can use the skills that they have learned in Biotechnology 1015, and
apply them in a real world situation. The mystery plasmid that was given to the students
is going to be digested and run through an agerose gel electrophoresis (which separates
based on mass) to help determine the plasmid. A longer incubation period was used so
that the enzymes could work to there full potential.
MethodsThe plasmid NA has a concentration of 17.4 µg/mL. The process that the
concentration of a plasmid is calculated is through spectrophotometery. Absorbance can
also be calculated through Beer’s law, A=C/ε. The DNA ladder is the 1Kb ladder from
New England Biolabs. The source of the enzymes and the buffer solution was from New
England Biolads. The digestion recipe 11.5µl of NA, 1.5 µl of NEB 4 buffer solution,
1µL of Dral, and 1µl of dH20, the total volume of the solution was 15µl. The second
double digest consisted of 11.5µL of NA, 1.5µl of NEB 4 buffer, 1µL of Dral, and 1µL
of BamHI. The NA digest incubated for 8 hours at a temperature of 37° degrees Celsius
(approximately 98.6° Fahrenheit). After the incubation of NA, the experiment called for
a 1% agerose gel to perform agerose gel electrophoresis (to examine the effect of the
digestion). This required a solution that called for 0.5 grams of agerose and 50mL 1x
TEA. It was then heated in a microwave for 30seconds, taken out and examined to check
if the agerose had completely dissolved, placed back into the microwave for two 15
second session and from there it was placed in ice for 15 seconds so that the beaker is
cool enough to grasp. After that it is pored into the sealed gel tray (with the comb placed
in the designated slot). Buffer that was used for both of the gels in the solutions was the
#4 buffer solution (provided by New England Biolabs). To predict the sizes of the
segments, the online wed tool NEBcutter was used to predict where and how large the
segment sizes should come out to. NEBcutter was located at the url,
“http//tools.neb.com/NEBcutter2/”. In this experiment the fragments where apparent
they all had very distinct marks, which indicates that the digest worked correctly.
Results and Conclusions-
The beginning plasmid had a concentration of 17.4µg/mL and the Plasmid NA
that was given is actually pBLU. Due to the connection between the sizes of the
segments, and how they related to the picture that was taken. But because the segments
didn’t quite line up with the estimated positions it make it nearly impossible to use
segment size to tell what NA was. The possible error that might have been made was that
our sample could have been mislabeled (this is possibly why the DNA ladder was in the
center of the gel). So greater precautions would be made to eliminate such errors from
occurring again in future labs.
ReferencesBOOK- Thieman, Palladino, A.C., 2009:Introduction to Biotechnology Second
Edition. Glossary G1-G18.
Website- New England Biolabs, 2011: NEBcutter. Internet:
<http://tools.neb.com/NEBcutter2/>.
3000
DraI Digest
2500
2413
2313
DNA Fragement Size (BP)
2000
1500
1000
692
500
0
14
Dral (mm)
16
Base Pairs
14.5mm
2413
15.5mm
2313
21mm
692
18
20
DIstance in MM
22
The blue diamonds in graph above represent
the distance that the DNA fragments
traveled(in millimeters. The line is a line of
best fit that links the points together in a
logarithmic manner.