Dna molecular weight marker xiv

Qualitative analysis of DNA fragmentation was performed by agarose gel electrophoresis of total genomic DNA extracted from untreated and allicin treated promastigotes. Leishmania promastigotes were exposed to allicin (30 μM and 60 μM) for 24, 48 and 72h. After drug exposure cells were washed twice in sterile PBS (1000 xg, 10 min, RT) and the total DNA was extracted from the cell pellet (10⁸ promastigotes) using the Apoptotic DNA ladder kit (Roche) following manufacturer’s protocol. The DNA was quantified at 260/280 nm using a NanoDrop ND-1000 spectrophotometer. The genomic DNA (5 μg) was run on a 2% agarose gel containing SYBR Safe DNA gel stain for 1 h at 100 V and visualized under UV light using the DNA Molecular Weight marker XIV (Roche).

28S ribosomal DNA (rDNA) was amplified by PCR using ReadyMix Taq PCR Mix with MgCl₂ (cat. n° P4600, Sigma) coupled with large-subunit of forward fluorescently labelled primer LSU1 (5′-GATAGCGMACAAGTAGAGTG-3′) and reverse primer LSU2 (5′-GTCCGTGTTTCAAGACGGG-3′) (Microsynth AG, Balgach, Switzerland) [32 (link)]. Extracted fungal DNA (5 μL), 1 μM (each) forward and reverse primers, and 25 μL of DNA polymerase reaction mixture were mixed with nuclease-free water to give a total reaction volume of 50 μL. The reaction mixture was incubated for 1 min at 94 °C; subjected to 30 cycles of 0.5 min at 94 °C, 0.5 min at 55 °C, and 0.5 min at 72 °C; and finally incubated for 10 min at 72 °C in an ABI 2720 thermocycler (Applied Biosystems, Inc., Carlsbad, CA, USA). Concentrations of the PCR products from the nail samples were estimated in 0.8% (wt/vol) agarose gels and the amount determined using DNA Molecular Weight Marker XIV (Roche) and ranged from no detection to 150 ng/μL.