3.3 Equivalence-Testing Algorithms
3.3.9 A (Nearly) Linear Algorithm, with Witness
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7. The tubes were allowed to stand for about 3 minutes in order to give time for the lysis to take place. After this, 10 ml of neutralization buffer was added to the solution and the tubes were inverted several times to allow for even mixture of the solution.
8. The neutralized lysate was added into the ZymoPURE syringe filter and this was clarified in a conical tube.
9. An aliquot of binding buffer was added to the cleared lysate in the conical tube and this was mixed thoroughly.
10. After this, the entire mixture in the conical tube was added into the Zymo-Spin V-P
183 APPENDIX VI
PROCEDURE FOR RE-SUSPENSION OF PRIMERS AND RE-CONSTITUTION OF PRIMERS WITH OTHER PCR REAGENTS AND TEMPLATE DNA SAMPLES FOR
MULTIPLEX PCR TECHNIQUE
1. The microcentrifuge tubes containing the forward and reverse primers were gently spinned in a centrifuge machine in order to bring the contents of the tubes to the bottom of the tube prior to re-suspension (Perez-Perez and Hanson, 2002).
2. To re-suspend the synthesized DNA primers for PCR technique, a given aliquot of nuclease-free water was aseptically added to each of the microcentrifuge tubes containing the forward and reverse primers for MBL genes and AmpC genes respectively (as synthesized by Inqaba Biotechnical Industries Ltd, South Africa), and as follows:
For blaIMP-1F: An aliquot of 616 µl of nuclease-free water was added.
For blaIMP-1R: An aliquot of 592 µl of nuclease-free water was added.
For blaIMP-2F: An aliquot of 641 µl of nuclease-free water was added.
For blaIMP-2R: An aliquot of 559 µl of nuclease-free water was added.
For blaVIM-1F: An aliquot of 549 µl of nuclease-free water was added.
For blaVIM-1R: An aliquot of 522 µl of nuclease-free water was added.
For blaVIM-2F: An aliquot of 648 µl of nuclease-free water was added.
For blaVIM-2R: An aliquot of 579 µl of nuclease-free water was added.
For MOXM-F: An aliquot of 576 µl of nuclease-free water was added.
For MOXM-R: An aliquot of 583 µl of nuclease-free water was added.
For DHAM-F: An aliquot of 701 µl of nuclease-free water was added.
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For DHAM-R: An aliquot of 576 µl of nuclease-free water was added.
For ACCM-F: An aliquot of 610 µl of nuclease-free water was added.
For ACCM-R: An aliquot of 622 µl of nuclease-free water was added.
For FOXM-F: An aliquot of 518 µl of nuclease-free water was added.
For FOXM-R: An aliquot of 534 µl of nuclease-free water was added.
3. After the addition of the different aliquot of nuclease-free water to each of the microcentrifuge tube(s) containing the respective forward and reverse primers for the MBL genes and AmpC genes as elaborated above, the tubes were carefully corked and shaken vigorously to ensure even mixture of the primers and the nuclease-free water.
4. After this, the microcentrifuge tubes containing the primers were placed on the test tube rack to allow for the dissolution of the primers in the nuclease-free water. The tubes were allowed overnight at ambient temperature in the refrigerator until further use. This mix served as the source of the forward and reverse primers that was used for the multiplex PCR technique (Perez-Perez and Hanson, 2002).
5. To carry out the multiplex PCR amplification of MBL genes in the test isolates, a final volume of 26.5 µl reaction mixture comprising: 0.2 µl of Taq polymerase enzyme U/µl, 2.5 µl of 10X PCR buffer, 2.5 µl of MgCl2, 1 µl of 10 pM from each of the forward and reverse primers, 2.5 µl of dNTPs, 3 µl of DNA template (from the test isolates), 14.8 µl of nuclease –free water. This was the master mix reaction used for the multiplex PCR amplification of MBL genes.
6. 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)
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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 95oC for 2 mins, and this was followed by 25 cycles of DNA denaturation at 95oC for 30 sec. The primer annealing was carried out at 48oC for 30 sec, and primer extension was carried out at 72oC for 30 sec. After the last cycle, a final extension step was carried out at 72oC 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 94oC for 3 mins, and this was followed by 25 cycles of DNA denaturation at 94oC for 30 secs. The primer annealing was carried out at 64oC for 30 secs, and primer extension was carried out at 72oC for 1 min. After the last cycle, a final extension step was carried out at 72oC for 7 mins.