Identifying a Plasmid

Victoria Udy-1Th
Plasmid Code: 3825B15
A plasmid is a piece of circular DNA that is found in bacteria separately from the
Chromosomal DNA. Most of the time, plasmids are used as biological tools to manipulate
genes and cause them to transfer over as well as express themselves.
(http://www.nature.com/scitable/definition/plasmidplasmids28). In order to allow us to
recognise a specific enzyme, we have to use restriction enzymes to cut certain points in the
plasmid so that we have different predictable fragments sizes. Once we have our plasmid
cut we need a way to lay out the fragments in order of size so to do this we use Gel
Electrophoresis. This is a mechanism where we insert the DNA into an agarose gel then
we attach electrodes and run the DNA towards the red electrode. Because DNA is polar,
the DNA travels only as far as it possibly can depending upon its size. Once it runs all the
way to the other end, we take the gel out of the electrode box and place it under UV light
within the UV imager, which causes the fragments to glow and we can observe and
measure the sizes of these fragments and from there conclude which plasmid it is that we
are testing. This is the method I used along with the use of excel to graph my subsequent
fragment sizes. If we are able to successfully figure what plasmid we have, it can be shown
that we can use all of the knowledge we have gathered this semester and are able to use it
inaBiotechnologyworksetting.
The plasmid I was trying to identify had a codename of 3825B15. We obtained the
Plasmids, Restriction Enzymes, and Ladders from New England Biolabs. The first thing I
did was figure which restriction enzymes I should use. For this I used the program
NEBcutter (http://tools.neb.com/NEBcutter2/) and to get the sequences, I used
(tinyurl.com/plasmidseq). I decided to use BglI for my single digest, and Ndel and Pstl for
my double digest. Once I knew which enzymes I could use to properly identify my plasmid I
found the concentration of my plasmid was 250 ng I was then able to do some dimensional
analysis and figure that since my miniprep was 250 ng, I should use 2l of my miniprep.
Because I now knew the enzymes I was going to use, I looked at a chart
(tinyurl.com/nebdoubledigest), (tinyurl.com/nebbuffers) to figure which buffers would work
best with those enzymes. Here I found that for both my single and double digest I was going
to use Buffer 3. Then, because that we were using 10X Buffers I was able to find that I was
going to use 2l of Buffer 3. I then brought these DNA mixtures to a volume of 20l total
using dH
2
O. From here I flicked the tubes and set them to incubate for exactly one hour.
While these were incubating, I created my 8% agarose gel by measuring out 0.48g of
agarose powder and adding 60 ml of 1X TAE. To make this is used 6ml of our stock
solution of a 1X TAE and brought to volume of 60 ml. I then put this in a flask with the
agarose gel and placed it in the microwave for 45 seconds and then 15 second intervals
until the agarose was completely melted. I then added 1l of Ethidium Bromide to my liquid
gel, to cause the DNA to be able to mutate and run through the gel properly. Once the gel
cooled to the point where I could touch it with my bare hand, I was able to pour it into a gel
tray mold and then I placed a 10 well comb on one end of the gel. I let the gel set up until my
plasmid mixture was done incubating. I then added to my plasmids 4l of a 6x loading dye
as well as 5l of 1 kb ladder. I then filled the gel box with 280 ml of 1x TAE from which I
diluted by adding 28 ml of a 10x TAE and brought to volume of 280 ml. I then loaded my
DNAintomygelasfollows.
Ladder Control Bgll N&P Ladder
I then allowed my gel to run at 140 V for approximately 40 minutes, until the dye had
traveled between the 4 and 5 marker on the gel tray. I then detached the electrodes from
the gel box and removed the gel from the box entirely. I then carried it over to the UV
imager and made adjustments to clarify the image and took two pictures of differing
resolutions to ensure clarity of every band. However, before I ran the gel I calculated what
thebandsshouldlooklikeforeachpossibleplasmidasfollows:
FragmentsizeforBgll Fragment size for Ndel &
Pstl
pAMP 3263,1118,158 3134,1405
pKAN 3139,795,261 2635,923,636
pBLU 2121,1740,1576 2998,1316,926,197

Once I had
the images of my
gel, I was able to
measure the
distance that each
of the bands
traveled. Then I
was able to make
a standard curve
line on excel to find that my R
2
= 0.997 for the

exponential curve. The equation was
y=77607e
0.101x
From here I input this equation
into my chart and figured the fragment sizes of
all the bands from the control, Bgll digest and
Ndel&Pstl.Thisisshowninthechartbelow.

Control Bgll Ndel&Pstl

12600 4233 3638
8584 3389 508
5233 745
4452
As I observed this data more I came to realize that there was nothing that was very
conclusive within this set of data. My best guess was that I probably had pKAN, however,
the data was rather inconclusive and so I decided to do another run to try and accumulate
some solid data. This time I used the same procedures, except I did only single digests
and the enzymes were: SacI, Pvul, Pstl, and XbaI. In addition, I used CS Buffer on SacI and
Xbai and Buffer 3 on Pvul and Pstl. And I also allowed the enzymes to digest my DNA for 5
hours to ensure the DNA digested as much as possible. I then took pictures at three
differentbrightnessestoensureclarityofeveryband.
Iloadedthewellsasfollows:
Ladder SacI Pvul PstI XbaI Control Ladder

Fromthis,Ipredictedthefollowingfragments:
pBLU pAMP pKAN
Pvul
1980,1798,726,480,450
3643,896 3622,572
SacI 3495,1942 NoCuts 4194
Pstl 3924,1316,197 4539 3271,923
XbaI 5437 NoCuts NoCuts

I then made a standard curve for this like and my R

2
= 0.9986 and the equation that I used

to find the size of the real fragments was y = 103864e
0.101x
. From this I got the following

chart:

Pvul Pstl XbaI SacI

3896 3384,
815
13503,
8835,
6734,
4168
13503,
8835,
6734,
4168
From this data I was able to conclude that my plasmid is indeed pKAN. I can see this by
comparing the chart of the expected fragments with the actual fragment sizes. They are
very closely comparable except for the fact that my SacI did not digest. This is an
acceptable flaw because based on the two that did digest correctly, I can conclude the
sizes of the bands to have correctly digested for pKAN. Also, because my standard curve
isnt very tight so the accuracy should be within the 500 and all of the fragments were well
withinthatrange.