The DNA from the pooled mosquitoes was extracted by a GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, USA), according to the manufacturer’s instructions. Detection of microfilaria was performed using a specific primer for B. malayi (Hha1 F 5′-GCG CAT AAA TTC ATC AGC-3′, Hha1 R 5′-GCG CAA AAC TTA ATT ACA AAA GC-3′, AIT biotech) [16 ]. PCR was conducted in 50 ml reaction mixtures with the master mix reagents for the PCR, which consisted of Taq DNA polymerase (0.05 U/mL), reaction buffer (4 mM MgCl2), dNTPs (0.4 mM each), and oligonucleotide primers (0.1 mM each). The thermal cycling conditions were 35 cycles at 95°C for 3 min followed by 95°C for 30 min, 57°C for 30 s, 72°C for 1 min, and a final extension step at 72°C for 1 min. PCR products were separated by electrophoresis on a 2% agarose gel, detected by SYBR staining, and visualized with a UVITEC Cambridge Gel Documentation system. DNA was determined to be microfilaria positive in our previous study served as positive control.
Cambridge gel documentation system
Manufactured by Uvitec
Sourced in United Kingdom
The Cambridge Gel Documentation system is a lab equipment used for the visualization and analysis of nucleic acid and protein samples separated by gel electrophoresis. It provides high-quality imaging and documentation of gel-based experiments.
Automatically generated - may contain errors
2 protocols using cambridge gel documentation system
1
Mosquito Microfilaria Detection Protocol
2
Western Blot Analysis of IAP Protein
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SDS-PAGE-separated Caco-2 cell lysates were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) by electroblottting and non-specific binding sites were blocked [5% (w/v) BSA in Tris-buffered saline with 0.05% Tween 20 (TBS-T)] for 1 h, at 25 C (room temperature). The membrane was then probed overnight (18 h) at 4 C with a mouse monoclonal anti-human IAP Ab (diluted 1/500), followed by HRP-conjugated goat anti-mouse IgG (diluted 1:5000) as secondary Ab. The Abs were diluted in 2% (w/v) BSA in TBS-T. The membrane was developed with a chemiluminescent substrate (ECL Plus Pierce Blotting Substrate; Thermo Fisher Scientific) and the blot photographed using a UVITEC Cambridge Gel Documentation system (UVITEC, Cambridge, UK). Blots were stripped and re-probed with GAPDH Ab. For quantitative analysis, bands were detected and evaluated densitometrically using UVI band software (UVITEC), normalised for the density of GAPDH. 20
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