This study aimed to classify Mycobacteriophages isolated from Puerto Rican soil samples into clusters by analyzing their DNA using PCR and gel electrophoresis. The DNA of 8 phages (including Carmina, Phaglus, Maximus, and Suave) did not show clear results or bands, so their clusters could not be determined. The others were classified into clusters B2 or E based on the bands, except Fenixious which showed bands for both B2 and E, possibly due to mixed DNA samples. Errors in primer addition, reagent amounts, and sample mixing could have impacted the ambiguous results.

1. Cluster Classification of Mycobacteriophages Isolated from Tropical Soils of Puerto Rico.
Astrid Diaz¹, Nicole Colón¹
University of Puerto Rico, Cayey campus¹
Abstract: Mycobacteriophages have been studied through time for a number of reasons. They
have been used as model systems for the study of biological processes. This study aims to
examine and analyze these bacteriophages in order to classify themto a cluster. Each group of
researchers received a specific Mycobacteriophage. The bacteriophages’ DNA were amplified
using the PCR method, and were later analyzed by running an electrophoresis gel.
Introduction:
For many decades’bacteriophages have and form plaques . The plaques represent
been know asviruses that infect bacteria. the cycles of infection and cell lysis, which
Each Bacteriophage is composed of a head identify the phage. After identification, the
and a tail (figure 1.1). The head, stores the plaques are purified for further
genetic material, while the tail is used to characterization.
inject the genetic material into the host
bacterial cell. Phages can be classified For many years,
according to their morphology and nucleic Mycobacteriophages have been studied and
acids. The International Comittee on analyzed in order to understand biological
Taxonomy on Viruses has study and processes. Around 2,400
classifiedover 2,475 species in 2011. Mycobacteriophages have been analyzed
and characterized. “Over 70 universities and
collages around the United States, have
isolated, purified, and characterized
“Mycobacteriophages are viruses Mycobacteriophages from soil samples”
that infect bacteria belonging to the (Rubin 2012). This experiment analyzes and
mycobacteria genus”(Rubin 2012). examines a specific phage in order to assign
Mycobacteriophages can be found in a it to a cluster.
variety of soils. They can be classified as
harmless bacteria or disease causing agents.
The size of a phage depends onthe average
number of phage particles liberated when an D
infected bacterium is lysed.
Head
Bacteriophages can be isolated by,
Tail
enriching soil samples in a nutrient media
containing the bacterial host. Doing this will
let the phage reproduce, increase in number,
Figure 1.1 shows the Structure of a
bacteriophage.

2. Materials and Methods:
During this experiment a specific showed. Figure 1.3 contains the results of
Mycobacteriophage genomic DNA was the agarose gels. The bands of the
assigned. The genomic DNA designated was Mycobacteriophages indicate that they were
from Carmina Mycobacteriophage. The from a specific cluster. The thicker upper
preparation of Carmina’s DNA and the bands are bright, because they have the
primer were previously made. Test tubes genome, and the ones that ran towards the
were labeled from 1 to 15. To each tube a bottom of the wells are the primer
certain amount of each reagent was added. replications of a specific region of the
First, 5 l of Nano Pure PCR Grade Water genome As shown in the results, all the
(H2O) was added. Later, 5 l of Carmina’s Mycobacteriophages belongs to a cluster,
genomic DNA was added to each tube. except three of them. These three (Carmina,
Following this step 1 l of the specific Phaglus, Maximus, Suave). Table 1.1
forward and reverse primers were also represents the specific clusters of the
incorporated. In addition, 12 l of the PCR Mycobacteriophages that were analyzed.
Master Mix, which contained Taq
Polymerase, Buffer, Nucleotides, Mg2+,
were added. The tubes were placed in a Figure 1.2shows the control gel.
thermocycler and amplified. Once
amplified, the electrophoresis method was
performed. The agarose gel was previously
prepared. A variation of the electrophoresis
procedure was carried out. Instead of adding
2 l of loading dye, 4 l were added. Also,
only 8 l of the reagent of the PCR reaction
was used not 10 l. (Explain why the
original formula was altered.) The wells of
the 2% agarose gel were loaded and they
were left to run for one hour and forty-five
minutes. This whole procedure was
repeated eight times with eight
Mycobacteriophages. Each group composed
of two lab partners was in charge of one of
the eight procedures.
Results:
The gel was analyzed and photographs were
taken using a gel documentation system.
Figure 1.2 shows the controls on an agarose
gel. In controls 11 and 15 the bands did not
Figure 1.3shows a photograph of the results
of the experimental gels.

3. This study provides information
about the classification of each
Mycobacteriophage. We cannot arrive at
conclusions because the results were not
clear.The Mycobacteriophage named
Carminahasambiguous results, because it
did not show any bands. One reason for this
could be that the investigator made an error
while adding the primers to the test tubes.
The tubes labeled from 1 to 8, had A1
forward and reverse primer. This may have
affected the reaction and results. Also, in
Agarose gel #4, nothing showed, not even at
Table 1.1shows the specific clusters the controls. A reason for this may have
belonging to each Mycobacteriophage. been incorporating incorrect amounts of
reagents in the tubes. Another error may
Mycobacteriophage have been that primers were mixed up.
There is an ambiguity with the cluster
Cluster named Fenixious. In the agarose gel two
bands, in different wells, are present. This
Bruce B2 Primers shows that it belongs to the E cluster but it
also has a band in the B2. A possible reason
Carmina Results are not for this may be that since the samples are
clear taken from the soil and they containmany
Mycobacteriophages, there may have been
Cemi B2 Primers more than one DNA present. This could
have happened during the purification of the
Fenixious E Primer DNA. Also, the investigator may have
(thicker band), accidentally blended those samples.
B2 primer
(lighter band)
Acknowledgements:
Lorenzoveg B2 Primer
Special thanks to: our mentor Dr.Rubin, the
NovaAndreas B2 Primer TA’s, Melisa Medina and Valeria Rivera,
Yadira Ortiz and RISE. Also to Eneida Díaz,
Phaglus_Maximus Results are not and Elena González.
clear
Suave Results are not
References:
clear
- Rubin. M. 2012. Cluster Classification of
Mycobacteriophages Isolated from
Tropical Soils of Puerto Rico.
Discussion: