Aubrey Jones
Jean Bower
Biotech 1010-F13
Plasmid Report
What Are My Plasmids?
Introduction
What would happen to genes without plasmids? Well, without plasmids, they could not
transfer over to other DNA cells. Essentially, living organisms would have less DNA without
plasmids. On the other hand, restriction enzymes cut DNA nucleotides like scissors. They are
used to sequence the DNA to find out what DNA the sequence exactly is. How can we find out
which plasmids contain which restriction enzymes? Well, through gel electrophoresis, we can
identify exactly where restriction enzymes cut the DNA. For the gel electrophoresis once we
place the restriction enzymes (digests) in a solidified agarose gel, we electrocute them to run
down the agarose gel. After about an hour, the agarose gel will accurately show how far the
restriction enzymes cut the DNA. The goal of the plasmid identification project is to determine
the plasmid according to our restriction enzymes with gel electrophoresis. This goal is
important because it will help us understand exactly what plasmid and eventually, the DNA
gene it is. To achieve my goal, I used the strategy of the gel electrophoresis and a standard
curve to identify my plasmid.
Methods
For my digest BgII, my plasmid was pAMP and for my digest EcoRI + HindIII, my plasmid
was pBLU. I was able to determine the concentration of my plasmid by plugging in the
equations y = 0.0046x + 0.2509 and R2 = 0.967 for Digest BgII and then y = 0.0026x + 5.1793
and R2 = 0.6301 for the double digest of EcoRI + HindIII. I found these equations through
creating a standard curve and clicking on one of the restriction enzyme points. The source of
my marker DNA was through a miniprep stock. I also created different solutions with the
sources of a Buffer R, BgII enzyme, and EcoRI + HindIII enzymes. There are three digest
recipes I created. First, was the control with three microliters of miniprep, two microliters of
Buffer R, and fifteen microliters of dH20. Next, I had the single BgII digest with three
microliters of miniprep, two microliters of a Buffer R, one microliter of BgII, and fourteen
microliters of dH2O. And last, I had a double digest of EcoRI + HindIII of three microliters of
miniprep, two microliters of Buffer R, one microliter of EcoRI, one microliter of HindIII and
thirteen microliters of dH2O. These digests incubated for 24 hrs at 37 degrees Celsius. After I
incubated the digests, I placed them in a solid agaorose gel of .8%. To make this agarose gel, I
measured out .32 g of agarose and poured it into a glass bottle. Measuring 40 mL of 1X TAE
into a 100 mL graduated cylinder, I mixed this into the glass bottle with the agarose. I
microwaved this agarose/1X TAE mixture for 45 seconds. I added in 1 microliter of ethidium
bromide solution to the gel mixture. Once this gel cooled, I electrocuted it through
electrophoresis at 130 volts for about an hour to allow the digest recipes to run. For my gel, I
used a Buffer R stock (previously made, not by me). In order to record my images of my gel, I
took a picture of it and created the standard curve for each the single and double digest. I
placed them in my notebook. I had determined my fragment sizes by performing virtual digests
on the computer and compared them with the ones in my picture. In order to compare them, I
used a ruler to know the distance traveled by in mm on the agarose gel. This information on the
standard curves allowed me to predict my digest results.
Results and Conclusions
Plasmid Identification Project Gel
As I was not supposed to add in a DNA Ladder, at the top are the distances traveled from in
mm for the three lanes of Control, BgII, and EcoRI + HindIII. I also will add in a few more
here to match my standard curves. From what we can see, the Control lane has 15 mm and 10
mm. BgII has 16 mm, 14 mm, and 10 mm. And EcoRi + HindIII has 18 mm, 17 mm, 12 mm,
11 mm, and 10 mm.
Y = 0.0046x + 0.2509 and R2 = 0.967
Y = 0.0026x + 5.1793 and R2 = 0.6301
This is a table compiling of all of my restriction enzyme single and double digests expected
fragment sizes for plasmids of pAMP, pKAN, and pBLU
Actual Fragment Sizes (bp) for Single and Double Digests that I Could See on my Agarose Gel
Picture are below:
BgII Single Digest
pAMP
pKAN
pBLU
3263
3139
2121, 1740, 1576
EcoRI + HindIII Double
Digest
2635, 1904
2312, 1882
5386
Because of my actual sizes, I know that my BgII single digest is a pAMP plasmid because it
has the highest fragment size marking. In other words, the BgII runs farther and becomes
pAMP. Now for EcoRI + HindIII, it has the pBLU plasmid for it is a fragment size of 5386
compared to the much shorter fragments of pKan and PAMP.
References:
“Plasmid.” Dictionary.com Dictionary.com, n.d. Web. 4 Dec. 2013.
http://dictionary.reference.com.
“Restriction Enzymes.” Dictionary.com. Dictionary.com, n.d. Web. 4 Dec. 2013.
<http://dictionary.reference.com/browse/restriction+enzymes?s=t>.
“Plasmid Identification Project” Biotech 1015-001-F13 n.d. Salt Lake Community College 4
Dec. 2013
“Gel Electrophoresis Activity” Biotech 1015-001-F13 n.d. Salt Lake Community College 4
Dec. 2013
“Standard Curve Exercises” Biotech 1015-001-F13 n.d. Salt Lake Community College 4 Dec.
2013