Cloning DNA with Plasmids

Makayla Claiborne
Abstract:
Using cloning allows us to do so many thing in science, like replicate crime scene DNA
to stem cells, but potentially anything with DNA. To allow us to replicate the sample properly we
must use restriction enzymes to tell us about the DNA since we are not able to see it by looking
at it.
Introduction:
In this experiment we are using restriction enzymes to cut the DNA, and ligation to add
in other strands of DNA. We transform the sample into a vector, EcoRI, onto Kan plates. The
reaction should have a Kan resistant gene added to it (via ligation and restriction enzymes), and
the control does not. If the bacteria successfully grows, then the reaction works, and we proceed
to isolate the DNA from the bacterial colonies. After we purify and isolate the DNA here we can
use different restriction enzymes to tell us about our sample by running gel electrophoresis.
Methods:
Our class used the Edvotek 301 standard cloning and transformation protocol to conduct
this lab. The basic steps of the protocol are outlined in the five modules below.
In module one our group produced a ligation control sample (C1), a ligation reaction
sample (R1), and a DNA ligase tube, or T4, (used for module 2). We centrifuged the stock
ligation mixture in the tubes, and incubated the samples in a water bath at 16 degrees C. After the
samples were properly mixed, we pipetted our standard marker (lane one), C1 (lane 2), and R1
(lane 3) in agarose gel. Then we performed gel electrophoresis, and stained our gel. After
staining and de-staining the gel, we compared the bands in each lane using a white light
visualization system.
In Module two, we created two new samples, a ligation reaction (R2), and a control
reaction (C2) using T4 from module one. C2 is transferred to a control kanamycin plate, and R2

is added to the ligation kanamycin plate. The plates are then incubated overnight to allow
bacterial growth to occur.
Module three consisted of mixing the growth of EcoRI bacteria on the ligation plate from
module two with the liquid Kanamycin, and incubated in a shaking water bath overnight.
Module four has us take pellets from the EcoRI, from the sample made in module 3, and
add buffers to the mixture. We allow the sample to separate into supernatant and a pellet by
centrifuging the sample. We pour out the supernatant, add ethanol, and spin again to get rid of
more supernatant. Once we isolate the pellet, we add buffer, and put the sample on ice.
In module five, we created a restriction digest cocktail (RDC) and added it to four
separate tubes along with corresponding restriction enzymes. After incubating, the samples are to
be loaded in agarose gel. Lane one contains the DNA marker, lane two is loaded with a
supercoiled plasmid vector, and lanes three through six contain the mixtures of the RDC made
earlier in this module. Lane three is the control restriction digest (no added enzymes), lane four is
EcoRI digest, lane 5 PvuII digest, and lane 6 is the double digest of PvuII and ClaI. The gel was
run through gel electrophoresis. After the samples were run long enough, the gel was stained and
de-stained. The finished gel was viewed on the white light box to view the bands.
Results:

Module 1:
Although the bands in the ladder in lane one did not show up to well, the ligation control sample
in lane two, and the ligation reaction sample in lane three proved that the reaction was a success.

Modules 2:
The colony growth on the kanamycin plate in the picture above indicates that the Kanamyacin
resistant gene in module one was successfully inserted into the bacteria.

Module 3:
After we mixed colonies from our plates in module two into a tube containing Kanamyacin
broth, the tubes were shaken in a water bath overnight.

Module 4:
The pellet in the tube, located in the red circle, contains the wanted DNA.

Module 5:
Lane four containing the EcoRI digest, and lane six containing the double digest both only had
one band instead of two (or possibly three for lane six) making the gel electrophoresis
unsuccessful.

Discussion:
In module one, our gel models a successful gel electrophoresis of our control and reaction
ligation samples. The DNA marker did not show up as perfectly as we wanted it to. The control
produced two bands approximately 3,000 bp and 1,300 bp, and the reaction ligation had one
larger band of around 10,000 bp. The first time doing module two, we had no success in growth
on our ligation plate, but we had two small colonies on the control. This meant somewhere along
the way we were not careful and contaminated our control. The cultures of EcoRI were
successfully mixed in with the Kanamycin medium in module three. We kept a DNA pellet
through the end of the sample and successfully resuspended the pellet with buffer during module
four. Our gel from module five did not have bands resembling a successful digestion of the
Kanamycin resistant gene, which could be for numerous reasons like the gene was not digested,
or the DNA was not loaded correctly into the gel. As a group we could have been more careful
with our steps and with our septic technique. In doing this, we probably would have had success
with module two on the first try.
The fragments in each lane on our gel should tell us that each restriction enzyme was
digested. Lane one contains our DNA marker that we will be using to compare the length of the
other fragments in the other lanes. In lane two we have our supercoiled plasmid vector. We
expect to have several fragments. The orientation of all fragments in all lanes will depend on
how each the DNA inserted. Lane three has the restriction digest control, and should have several
bands. The EcoRI, lane four, should have two bands, one larger one at 3,000 bp and a smaller
one at around 1,300 base pairs no matter the orientation it takes. Our PVUII digest is in lane five,
and should have one 4,300 bp long band no matter what orientation the sample takes. The last
lane, lane six, is our double digest sample with PVUII and ClaI. Depending on what orientation
it takes, we should expect two or three bands with varying lengths for each orientation.

In doing this experiment we placed a specific DNA inside of a segment and ran it through
a gel and streaked it on a plate to see if it was successful. Some scientists do this in order to
transfer certain genes to plasmids, such as resistance to certain diseases. Using a gel or plate
allows us to make sure the ligation was successful before further replicating the new plasmid.
Once we know the new plasmid is successfully ligated, we can further replicate it. This process
is used for bioengineering new DNA.

Citations:

Addgene. (n.d.). DNA Ligation. [Accessed 20 Feb. 2016]. Retrieved from:

https://www.addgene.org/plasmid-protocols/dna-ligation/

Addgene. (n.d.). Plasmid Cloning by Restriction Enzyme Digest (aka Subcloning).

[Accessed 20 Feb. 2016]. Retrieved from: https://www.addgene.org/plasmidprotocols/subcloning/

Biotechnology Learning Hub. (28 Nov. 2007). DNA Cloning. [Accessed 20 Feb.
2016]. Retrieved from: http://biotechlearn.org.nz/themes/dna_lab/dna_cloning