Testing Equivalence in JFLAP

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chamber of the thermal cycler machine and the multiplex PCR program with the right PCR conditions were set and the process was allowed to run.

7. The initial denaturation temperature (for MBL gene amplification) was at 95^oC for 2 mins, and this was followed by 25 cycles of DNA denaturation at 95^oC for 30 sec. The primer annealing was carried out at 48^oC for 30 sec, and primer extension was carried out at 72^oC for 30 sec. After the last cycle, a final extension step was carried out at 72^oC for 2 mins.

8. To carry out the multiplex PCR amplification of AmpC genes in the test isolates, a final volume of 26.5 µl of the master mix comprising: 0.2 µl of Taq polymerase enzyme U/µl, 2.5 µl of 10X PCR buffer along with 2.5 µl MgCl2, 1 µl of 10 pM from each of the forward and reverse primers, 2.5 µl of dNTPs MIX (2 Mm), 3 µl of DNA template (from the test isolates), and 14.8 µl of nuclease-free water was used. This was the master mix reaction used for the multiplex PCR amplification of AmpC genes.

9. The master mix was properly vortexed in a vortexer prior to the gene amplification process in the thermal cycler. The Eppendorf tube(s) were placed in the heating (block) chamber of the thermal cycler machine and the multiplex PCR program with the right PCR conditions were set and the process was allowed to run.

10. The initial denaturation temperature (for AmpC gene amplification) was at 94^oC for 3 mins, and this was followed by 25 cycles of DNA denaturation at 94^oC for 30 secs. The primer annealing was carried out at 64^oC for 30 secs, and primer extension was carried out at 72^oC for 1 min. After the last cycle, a final extension step was carried out at 72^oC for 7 mins.

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APPENDIX VII

PROTOCOL FOR PREPARATION OF 1.5 % AGAROSE GEL

1. To prepare 1.5 % agarose gel, 3 g of agarose powder and 200 ml of Tris boric acid ethylene diamine tetraacetic acid (TBE) buffer solution is required (Perez-Perez and Hanson, 2002).

2. A volume of 200 ml of TBE buffer solution was measured using a clean measuring cylinder and this was dispensed into a clean/sterile beaker.

3. Three (3) gram (g) of the agarose powder was measured out using an analytical balance and a weighing paper.

4. The measured agarose powder was dispensed into a clean conical flask and the 200 ml of TBE buffer solution was added to it.

5. The conical flask containing the agarose and the TBE buffer solution was swirled vigorously to ensure even mixture of the agarose solution.

6. After swirling, the conical flask containing the solution was heated to boil for one minute in a microwave oven. The heating was allowed to continue until the solution became clear, and swirling of the solution was intermittently carried out during the heating process.

7. The heating was stopped once the agarose starts to boil to avoid over spilling in the microwave, and the conical flask was swirled until the mixture was clear. If the agarose was not dissolved completely, the finished gel will have regions of different concentrations; and this will make the nucleic acid samples not to separate correctly.

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8. The heated agarose gel was allowed to cool to about 60^oC before pouring the molten gel into the gel casting chamber. Hot gel damages the gel casting box or tray. The heated or hot gel was placed in a water bath regulated at 60^oC.

9. The gel tray was placed into the gel casting chamber and appropriate casting comb(s) was inserted into the chamber. The casting combs creates cavities known as wells in the gel, and it is into these wells that the DNA samples was pipetted into.

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APPENDIX VIII

PROTOCOL FOR GEL ELECTROPHORESIS TECHNIQUE

1. An aliquot of 6 µl of ethidium bromide dye was added to the molten gel prior to pouring.

Ethidium bromide is a mutagen and a possible carcinogen that absorbs into the skin and could be breathed into the body via the respiratory tract. And thus, hand gloves should be used and changed once ethidium bromide dye is handled and nose mask should also be used to avoid contamination.

2. The conical flask containing the molten agarose gel and the ethidium bromide was swirled vigorously to ensure even mixture of the gel and the dye.

3. Molten agarose gel was poured into the gel casting chamber to a depth of about 5-7 mm with the casting comb in place. All bubbles formed during this process were gently removed using the ends of a pipetting tip. Bubble formation prevents free flow of electric current through the gel during the electrophoresis process.

4. The molten gel was allowed for about 20-30 minutes to solidify. The poured gel was allowed to cool in the gel chamber or tray and when its appearance was opaque, it was ready for the next stage. Usually, the colour of the gel changes from colourless to slightly opaque. While the gel is setting, it is important not to move the gel tray or disturb the gel itself – since this could create gel of non-uniform fitness.

5. After solidification or setting, the casting combs were removed carefully by pulling it upwards firmly and in a smooth and continuous motion. Some resistance is usually felt as the casting comb was removed from the solidified gel. Ignore it and gently remove the comb in an upward position as earlier stated (Perez-Perez and Hanson, 2002).

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6. The set gel contained in a gel tray was transferred into a gel rig while ensuring that the wells on the cast gel was on the cathode (left) side of the gel rig.

7. The gel rig containing the gel was filled with 0.5 X TBE buffer solutions until the gel was entirely covered by it. Usually, the buffer solution should be above the surface of the wells in the gel at a distance of about 2 mm.

8. The gel chamber or rig was placed on the surface of a dark background in order to easily visualize or see the wells during loading of the DNA samples. The dark background helps you to see the wells when adding the DNA samples.

9. Before loading the DNA samples to be analyzed into the wells of the gel, a coloured loading buffer (5 X bromophenol blue with ficole) was added to each of the DNA samples in a microcentrifuge tube. The coloured loading buffer is made up of dye and a glycerol of ficole solution. The dye colour helps to visualize the loading of the samples into the wells and it also helps to keep track of the migrating DNA samples in the gel while the glycerol or ficole makes the DNA samples heavy so that they sink to the bottom of the wells.

10. The coloured loading buffer was mixed properly with the DNA samples in a