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Abstract: The procedure described in this lab report took place over the course of several lab
periods. Fusarium vertilloides DNA was digested to completion, inserted into a pUC-19 plasmid,
which in turn was used to transform E. coli. The competent cells were then plated and after it
was determined which colonies had successfully taken up the plasmid, pUC-19 was extracted
and a random primed synthesis and a southern blot conducted, according to Biotin-Streptavidin
labeling methods. This enabled analysis of the size of the digested DNA fragments and their
molecular weight.
Introduction: Southern Blot Analysis is an important way to detect either the action of genes
within certain cells or, in this case, the action of different restriction sites within genomic DNA.
In combination with the methods used in this series of protocols, ranging from bacterial
transformation of E. coli, to random primed synthesis, southern blot may be a valuable tool.
Final analysis of the membrane produced by a southern blot which has been conducted on
digested genomic DNA can lead to conclusions on the molecular weight of fragments,
approximate cutting locations, and other things of interest.
Materials and methods: DNA was initially isolated from a fungus, Fusarium verticilloides,
using Exercise 5 from the lab manual for Techniques in Recombinant DNA (Jurgenson). The
DNA was stored at -20C for several weeks until it was used in this protocol.
To create hybrid DNA molecules, the F. verticilloides genomic DNA was first cut with
different combinations of restriction endonucleases in a solution of pre-made buffer and water.
The specific enzymes used on this sample were Eco-R1 and Bam-H1. The samples were allowed
to digest to completion, incubated overnight at 37C. After digestion, digested DNA was
concentrated using the speed vacuum concentrator to a volume of about 15 l.
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The samples were run on a 0.7% agarose gel containing guanosine for 20 minutes at 200
volts. Guanosine was used in order to prevent degradation of the DNA by ultraviolet light, so it
is suitable to use in downstream applications. Image 1 shows a picture of this gel, with a Hind III
ladder. In addition to the F. verticilloides digest, pUC-19 plasmid vectors were also digested
with Eco-R1 and Bam-H1, both of which have cutting locations in the multiple cloning site. This
was the plasmid to which the yeast genomic DNA would be cloned into.


The desired bands of DNA were cut out of the agarose gel and using kit the gel was
dissolved and DNA isolated. This protocol began with 0.1 g of gel square with the selected bands
being added to a microcentrifuge tube. 500 l of DF buffer was added and the tube was
incubated at 55C for 15 minutes and inverted every 3 minutes. The sample was allowed to cool
to room temperature and then 800 l was placed in the DF column, which in turn was placed in
the 2 ml collection tube. This was centrifuged for 30s at 15000 rpm and the pass-through was
Figure 1
Restriction digest of genomic DNA
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discarded. 400 l of Wash 1 (W1) buffer was added to the column unit and it was again
centrifuged for 30s at 15000 rpm, and the resulting pass-through was discarded. Then 600l of
W1 buffer was added to the DF column and it was left to stand for 1 min. Again it was
centrifuged at 15000 rpm for 30s and the pass-through was discarded. In order to dry the glass-
bound DNA, it was centrifuged for 3 min at 15000 rpm. The dry column was then transferred to
a new 1.5 ml microcentrifuge tube and 30 ml of water was added to the column matrix and left to
stand for 2 min. It was centrifuged one final time for 2 min at 15000 rpm, and then the pass-
through, containing the digested genomic DNA was frozen for use next lab period.
At this point, the previous restriction digests had not turned out well, and Dr. Jurgenson
provided a new set of digested F. verticilloides DNA, which was then purified according to the
same procedure outlined above.
The mixes for DNA ligation were prepared consisting of 3,4, or 5 l plasmid, 1 l 10x
ligation buffer, 0.5 l T
1
ligase, and 4.5 l of the genomic DNA. The ligation mix and 5 l T.E.
were added to a transformation tube and set to chill on ice. The competent E. coli cells were put
on ice and 7 l of DTT was added. 200 l of the competent cell mixture was added to the chilled
transformation tube containing the digested genomic DNA, and it was gently agitated to ensure
mixing of the DNA and cells. This was left to chill for 30 min, then a heat shock was performed
at 42 C for 90s, then immediately the tube was set on ice for 90s. 800 l of SOC was added and
the transformed cells were incubated at 37 C at 225 rpm for 1 hr, in order to allow the
transformants to produce amp
r
protein before they were plated on medium containing ampicillin.
After this, the 180 l of the cells were plated on each of 5 plates per ligation mixture.
They were allowed to incubate for 24 hours, at which point Dr. Jurgenson halted their growth by
placing them in the fridge. Analysis of the transformants indicated 10 samples which were then
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placed on a single plate. The dish was incubated in a 37 C oven to allow growth. Each of the
transformed colonies was also transferred to a test tube containing LB broth, allowing them to
multiply.
The next period, further analysis of the transformant colonies was performed. 1.5 ml of
each bacterial culture was placed in microcentrifuge tubes and spun for 1 min at 14000rpm. A
pellet was formed and the medium was removed, leaving the pellet as dry as possible. The pellet
was then resuspended by vortexing in 100 l of a solution containing glucose, EDTA, Tris HCL
ph 8.0, and water (Solution 1). The tubes were then stored for 5 min at room temperature. 100 l
of ice cold solution containing 1% SDS, 0.2 N NaOH, and water was added to the tube. It was
mixed by inversion and stored on ice for 5 min. After this, 150 l of ice cold potassium-acetate
pH 8.0 was added to each tube and they were gently vortexed for 10s and then iced for 5
minutes. These were then centrifuged for 5 min at 4 C at 10000 rpm, and the supernatant was
then transferred to a new tube. An equal volume of chloroform was added and the solution was
mixed by vortexing and centrifuged for 2 minutes at 10000rpm, and again the supernatant was
transferred to a new tube. 2 volumes of 95% ethanol was added and vortexed, then left to stand
for 2 min at room temperature. The test tube was then centrifuged for 5 min at room temperature
at 10000 rpm. Again, the supernatant was removed and the tube was stood in an inverted position
to allow extra fluid to drain away. 1ml of 70% ethanol was added, then poured off and the tube
was centrifuged. The supernatant was removed, and the sample was dried briefly in a 42 C
oven. Then 50 l of TE pH 8.0 with DNAse-free pancreatic RNA was added and the sample was
briefly vortexed.
The samples were run in the Speed-Vac concentrator and resuspended in 50 l of TE
with RNAse. A restriction digest was then performed with a solution containing DNA, enzyme,
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restriction digest buffer, and water. After complete digestion, the transformed pUC-19 plasmids
were run on a 0.7% agarose gel at 120 V for 40 minutes. Analyzing this gel, it was determined
which sample to use for our southern blot analysis.
Samples of the chosen pUC-19 plasmid were digested in class using different variations
of Bam-H1, Eco-R1, and Sal1, after electrophoresis, the gel was photographed with a ruler. That
is listed below in Figure 3. The gel was sliced up and a southern blot protocol was performed
according to Exercise 8 of the Recombinant DNA lab manual (Jurgenson). To begin, the gel
section was treated with 100 ml of 0.25 M HCl for 15 minutes. The gel was then rinsed with
distilled water, and 10 volumes of 0.4 M NaOh was added. The gel was soaked for 20 min with
slow back-and-forth tilting for 20 min.
The southern blot apparatus was then set up, with a stack of paper towels stacked 2.3 cm
high, 4 pieces of dry Whatman 3MM filter paper on top of them, and a wet piece of filter paper
placed on top of that. The positively charged membrane was then cut to be slightly larger than
the gel segment and wetted in 0.4 M NaOH solution. The membrane was placed on top of the
filter paper and it was made sure there were no bubbles between it and the paper. Plastic wrap
was placed around the membrane to ensure that the buffer did not transfer through the filter
paper. The gel was then placed directly on top of the membrane. 3 pieces of Whatman paper
were then soaked in 0.4 NaOH transfer buffer and placed on top of the gel. 2 long pieces of
paper were then soaked in transfer buffer and placed on top of the paper. One end of them was
then placed in the reservoir containing the transfer buffer. A weight was placed on top of the
paper towel stack to prevent evaporation and encourage downward transfer of the buffer through
the gel and membrane. The transfer was then allowed to take place overnight. After, the
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membrane was taken and rinsed in a 2x SSC solution and allowed to air dry in the dark on
Whatman 3MM paper.


Biotin labeling of the probe was conducted according to the Biotin Decalabel DNA
labeling protocol by Thermoscientific. 10 l of DNA template, 10 l of decanucleotide 5x
reaction buffer, and 34 l water was added to a 1.5 ml microcentrifuge tube. The tube was
vortexed, spun down, then incubated in a boiling water bath for 10 minutes, cooled on ice, and
spun down again. To the same tube was added 5 l Biotin Labeling mix and 1 l Klenow
fragment, exo
-
. The tube was shaken, then spun down again. The solution was incubated for 1 hr
at 37 C and the reaction was stopped by adding 1 l of 0.5 M EDTA pH 8.0.
Figure 2
Restriction digest of selected samples
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Labeling of the membrane with Biotin-labeled DNA was straightforward. In a sealed bag
was combined the positively charged membrane with DNA bound to it, a hybridization buffer,
the and the labeled probe. This bag was allowed to set at 65 C for 48 hours with moderate
shaking. After hybridization, the filter was washed to remove non-sepcific probe fragments with
SDS, and then the following procedure was conducted.
The detection of the Biotin-labeled fragments was done according to the protocol of the
Biotin Chromagenic Detection Kit by ThermoScientific. First, an excess of assay solution was
made consisting of Blocking/Washing Buffer (B/W Buffer), Blocking Solution, Diluted
Streptavidin-AP Conjugate, Detection Buffer, and Substrate Solution. The membrane was
washed in 30 ml of B/W Buffer for 30 min at room temperature with shaking. The B/W buffer
was then discarded, and 20 ml of Streptavidin-AP conjugate was added, then membrane was
incubated at 30 min at room temperature with shaking. The membrane was then washed by
incubating in 60 ml of B/W Buffer for 15 min, then this step was repeated with new buffer and
incubated in 20 ml of detection buffer for 10 minutes. The enzymatic reaction was then
performed by incubating the membrane in 10 ml of Substrate Solution. A picture of the labeled
membrane is included below in Figure 3.
Results: Along with the pictures of the gels produced during these procedures, perhaps the most
significant result obtained was the southern blot membrane itself. The bands on the membrane
indicate corresponding places on the gel where the genomic DNA is complementary to our
streptavidin-labeled probe. In addition, the membrane may direct further experiments concerning
investigation into the genome of F. verticilloides.

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Discussion: By examining this southern blot, it is possible to find the complementarity
sequences in the fungal genomic DNA in relation to their restriction sites. Then, if the location of
the restriction sites are known, it can be used to construct a map of the chromosome. From
Figure 2, and the corresponding picture of the gel slice removed from it for use in the southern
blot, it can be seen that all the bands on the membrane correspond to areas of the Hind III ladder
mostly between 2300bp and 4300bp. The bands in lanes 7 and 8 correspond to segments that are
significantly above 4300mp. Because it is possible to locate the cutting positions of the
restriction enzymes in the fungal DNA, and the sequence of hybridization is known, the
sequences in these bands can be placed within the genomic DNA. For example, lanes 1 through 6
1 2 3 4 5 6 7 8
Figure 3
(Bands marked by white line)
Southern Blot Membrane
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all appear to be in the same general range. They are fragments specifically within 2300bp to
4300bp. This means that there is a sequence complementary to the Biotin-labeled probe at
several places in the genome, from the restriction site, at varying distances between 2300bp,
3500bp, 2300bp, 2700bp, and 3000bp, respectively for lanes 1 through 6. Lanes 7 and 8 are
estimated to be roughly 6500bp from the restriction sites.
Overall, the most valuable thing this presented the class with was the chance to conduct
procedures that it is rare to do while still in school. The findings that resulted support the current
information that is available concerning southern blots and how they may be used. The results
from this series of protocols would definitely prompt further investigation into topics such as the
composition of the DNA, activity of purpose of certain genes, and how that may be used in the
future.
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Literature Cited
Jurgenson, J. (2013). Techniques in recombinant DNA, lab manual. Print.
ThermoScientific. (2012). Biotin chromogenic detection kit. In ThermoScientific (Ed.),
ThermoScientific. Print.
ThermoScientific. (2012). Biotin DecaLabel DNA Labeling Kit. In THermoScientific (Ed.),
ThermoScientific. Print.