SR-3F Test  Lahore 2023
Materials and MethodsDNA ExtractionGenomic DNA was extracted using the CTAB/SDS method or a commercial kit, assessed for integrity, purity, and concentration via 1% agarose gel electrophoresis.PCR AmplificationPCR was performed using region-specific primers for:16S rRNA V4 (bacteria)18S rRNA V4 (eukaryotes)ITS1 (fungi)The reaction included 25 l of 2x Premix Taq, 10 mM primers, 60 ng DNA, and nuclease-free water to 50 l. Amplification consisted of an initial denaturation at 94C for 5 min, followed by 30 cycles (94C, 30 sec; 52C, 30 sec; 72C, 30 sec), with a final extension at 72C for 10 min.Electrophoresis and PurificationPCR products were analyzed by 1% agarose gel electrophoresis, confirming expected band lengths. Products were pooled based on concentration and purified with the E.Z.N.A PCR Gel Extraction Kit.Library Preparation and SequencingThe library was prepared using the NEBNext Ultra-DNA Library Prep Kit for Illumina and sequenced on the Illumina Nova 6000 platform (PE250).