Embedding bacterial cells in agarose plugs prevents damage to large DNA fragments
from shear force and it is an important step in the preparation, treatment and
digestion of plugs for Pulsed-Field Gel Electrophoresis (PFGE). Embedding is
usually carried out using disposable or reusable commercial plug molds which
can be expensive to purchase and often physically and experimentally inflexible.
Removal of agarose blocks from small rigid molds can be cumbersome and challenging
and difficult to clean between uses. Plug molds custom-made from silicone tubing
(Burmeister and Ulanovsky, 1992) plastic syringes (Ho
and Monaco, 1995) rubber dental impression material (Van
Devanter et al., 1989) TygonR tubing, plastic drinking straw, glass
pipettes (Birren and Lai, 1993), transfer pipettes (Sheneman
and Katz, 2003) and acrylic molds (Herschleb et al.,
2007) have all been reported. In this study, an initiative has been taken
to transform the primer container covers obtained from First Base Malaysia into
PFGE plug molds and study the PFGE patterns generated by the preparations.
MATERIALS AND METHODS
The cover of a plastic primer container, from First Base (Malaysia), was found to be suitable to be used as a plug mold for present PFGE work. This fairly soft plastic cover consists of two rows of trough-shaped hollows with numbers 1 to 5 on the top row and numbers 6 to 10 on the bottom row (Fig. 1). The original and permanent numbering on the container is very useful for labeling the plugs. Commercially purchased reusable plug molds measure 15x
1 mm in size and are able to contain a volume of 150
56;L, which gives a plug thickness of 1 mm. Therefore, to prepare an equivalent thickness in the trough of our in-house mold, which is 50x
1 mm, we marked an area 15x
15 mm to contain 225
56;L of agarose cell suspension. The marking was done on the reverse side of the container for convenience and permanence. Both ends of each trough can be used to accommodate two samples at a time providing space for 20 samples in one primer container cover in the in-house plug mold (Fig. 1).
||Plastic primer container cover used in preparing 20
sample plugs for PFGE
DNA preparation, restriction, electrophoresis, staining and gel documentation
was carried out based on the method of Ribot et al.
(2006) with modification of the total volume of cell suspension and agarose
to 225 56;L including 10 56;g of Proteinase K (Promega). Digestion was carried
out with restriction enzyme NEB Xba1 concentration of 10 U 56;L- 1
per plug and overnight incubation at 37° C. A CHEF-DR II system (Bio-Rad
Laboratories, Munich, Germany) was used with the following parameters: initial
switch time of 5 sec, final switch time of 65 sec, voltage of 200, angle of
120° and run time of 28 h. Salmonella ser. Braenderup H9812 was included
as a molecular size standard. Reproducibility of the PFGE profiles was established
by repeated testing of the same preparations on separate occasions.
DNA restriction patterns were analyzed for similarity by Dice Co-efficient
and cluster analysis was performed by the unweighted pair group matching band
average (UPGMA) of Bionumerics Version 5.1 (Applied Maths, Belgium).
RESULTS AND DISCUSSION
Present foremost objective of this study was that the PFGE patterns generated
by preparations using commercial plug molds and the in-house plug molds should
be identical. This was found to be true when the PFGE profiles, generated by
the use of enzyme NEB Xba1, were analyzed by the Dice-coefficient of similarity,
F, which expresses the proportion of shared DNA fragments of 2 preparations.
In addition, the resolution and reproducibility of the band profiles were identical
and comparable (Fig. 2).
The dendrogram generated by the cluster analysis of the results show a profile similarity of 100% for Salmonella ser.
PFGE profiles using restriction enzyme-NEB Xba1. Lanes
1, 2, 19 and 20 are Salmonella ser. Braenderup H9812 universal standard.
The rest of 16 lanes odd numbered lanes are plugs prepared in commercial
molds and even numbered lanes plugs prepared in plastic primer container
Dendrogram of cluster analysis
(generated by Bionumerics
version 5.1 program using the UPGMA method), from plugs using commercial
and primer-container molds. Identical results indicate no difference between
preparations in either type of mold
Braenderup H9812, used a DNA size marker, regardless of the type of mold used. Identical results of similarity were also obtained with the other samples for both in house and commercial plug molds used (Fig. 3).
Generally, most laboratories do not purchase more than one commercial plug
mold due to cost and there are limits to the number of samples that each mold
can contain. With the in-house plug molds, using plastic primer container covers,
100 sample plugs can be prepared at the same time with just 5 of these covers.
Added advantages are the in-house molds are cheap, easily available, recyclable
and easy to clean and maintain unlike commercial molds which are usually square,
enclosed and unreachable due to it small and narrow size. The broad surface
and flat area in the mold promotes faster cooling and the agar sets quickly
at room temperature. Plugs are easily removed using a spatula leaving no residue
behind which makes cleaning easier and drying faster. It is not necessary to
use tapes or parafilms and no extra labeling is required as there are permanent
numbers on the covers. No clamping or cutting of the mold is needed to remove
the plugs.Overall, the easy to-use molds provide flexibility for producing different
sizes and thickness of plugs by controlling the volume of cells and agar to
be dispensed into the molds. They are also more user friendly compared to other
custom made, usually rigid plug molds with other materials, as reported by Burmeister
and Ulanovsky (1992), Ho and Monaco (1995), Van
Devanter et al. (1989), Birren and Lai (1993)
and Sheneman and Katz (2003).
Plastic primer container covers can be recycled and used as plug molds for pulsed-field electrophoresis. Researches who are working on PFGE can use this alternative method instead of using the expensive commercial plug molds.
We would like to thank Dr. Doinita Ispas from Bio-Rad Romania for advice, the Malaysian Civil Department and Ministry of Health for See Kah Hengs study leave and scholarship. This study was funded by eScience Fund from the Ministry of Science, Technology and Innovation, Malaysia (No. 12-02-03-2033).