Nested-PCR was carried out with total volume of 50 μL comprised of 25 μL of GoTag®G2 green master mix (Promega, Madison, WI, USA), 5 μL (1:50 dilution) RT-PCR product, 50 pmol (final concentration 1 μM) of each consensus forward primer NS2g and reverse primers NS3a (Table 1 ). Themal cycler was programmed for initial denaturation (94 °C × 3 min), 30 cycles denaturation (94 °C; 30 s), annealing temperature (55 °C; 30 s), primer extension (72 °C; 1 min) and final extension for 5 min. Nuclease-free water was used as negative control and RVFV cDNA of positive sample obtained from Saudi Public Health Authority Jazan was used as positive control. The nested PCR products were resolved by 2% agarose, stained with ethidium bromide and viewed by Gel Doc XR Imaging System (Bio-Rad, Irvine, CA, USA).
Gotag g2 green master mix
Manufactured by Promega
Sourced in United States
GoTag®G2 green master mix is a ready-to-use solution for performing quantitative real-time PCR (qPCR) with SYBR® Green detection. It contains all the necessary components, including a DNA polymerase, SYBR® Green dye, and buffer system, to amplify and detect target DNA sequences.
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Lab products found in correlation
4 protocols using gotag g2 green master mix
1
Nested-PCR for RVFV Detection
2
Molecular Identification of Culex Mosquitoes
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A 710 bp of template was amplified using the forward primer LCO1490 and reverse primer HCO2198 [21 (link)]. PCR mix with total volume of 25 μL comprised of 12.5 μL of GoTag®G2 green master mix (Promega) 2 μL (20 pmol) of universal forward and reverse primers, 2 μL of DNA template and 8.5 μL nuclease-free water. Thermal cycler was programmed for initial denaturation (94 °C × 3 min), 30 cycles [denaturation (94 °C × 60 s), annealing (50 °C × 60 s, extension (72 °C × 60 s)] and final extension (72 °C × 5 min).
In each run, we used nuclease-free water as negative control and extracted DNA from Culex pipiens mosquitoes reared in Saudi Public Health Authority insectary in Jazan as positive controls. The PCR products amplification analyzed by gel electrophoresis (1.5% agarose in Tris-Acetate EDTA buffer), stained with ethidium bromide and viewed by Gel Doc XR Imaging System (Bio-Rad, Irvine, CA, USA).
In each run, we used nuclease-free water as negative control and extracted DNA from Culex pipiens mosquitoes reared in Saudi Public Health Authority insectary in Jazan as positive controls. The PCR products amplification analyzed by gel electrophoresis (1.5% agarose in Tris-Acetate EDTA buffer), stained with ethidium bromide and viewed by Gel Doc XR Imaging System (Bio-Rad, Irvine, CA, USA).
3
Nested PCR for DNA Amplification
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In brief, primary and nested PCR were carried out in total 50 µl reaction volume, each containing 25 µl GoTag®G2 green master mix ready to use from Promega and 25 µM of each primer. Five µl of extracted DNA was used as a sample for the primary amplification and 2 µl of PCR product for the nested PCR. In each run, negative and positive controls were included. Thermal cycling was done in T100 thermal cycler (Bio-Rad, USA). Primers and PCR conditions are shown in Table 1 .
The PCR products of nested amplification were analysed by gel electrophoresis (1.5 agarose in Tris–Acetate-EDTA buffer) staining with ethidium bromide. The visualization was carried out using Gel Doc XR Imaging System (Bio-Rad).
The PCR products of nested amplification were analysed by gel electrophoresis (1.5 agarose in Tris–Acetate-EDTA buffer) staining with ethidium bromide. The visualization was carried out using Gel Doc XR Imaging System (Bio-Rad).
4
Mitochondrial DNA Barcoding using COI
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A pair of universal primers, LCOI490 (5-GGTCAACAAAT CATAAAGATATTGG-3) and HCO2198 (5-TAAACTTCAG GGTGA CCAAAAAATCA-3) (19) were used to amplify the standard COI-5 barcodes (i.e. c. 650-bp fragments at the 5-terminus of the mitochondrial gene for COI; Folmer standardized region).
The PCR was carried out in total volume of 25 μL containing 12.5 μL of GoTag®G2 green master mix ready to use from Promega, 2 μL (20 pmol) of each forward and reverse primers, 2 μL of DNA template and 8.5 μL nuclease free water. The mix was subjected to initial denaturation at 94°C for 3 minutes, 30 cycles of denaturation (94°C, 60 s), primer annealing (50°C, 60 s, primer extension (72°C, 60 s) and final extension for 5 minutes. In each run, negative and positive controls were included.
The PCR products amplification was analyzed by gel electrophoresis (1.5 Agarose in Tris-Acetate EDTA buffer) staining with ethidium bromide. The visualization was carried out using Gel Doc XR Imaging System (Bio-Rad).
The PCR was carried out in total volume of 25 μL containing 12.5 μL of GoTag®G2 green master mix ready to use from Promega, 2 μL (20 pmol) of each forward and reverse primers, 2 μL of DNA template and 8.5 μL nuclease free water. The mix was subjected to initial denaturation at 94°C for 3 minutes, 30 cycles of denaturation (94°C, 60 s), primer annealing (50°C, 60 s, primer extension (72°C, 60 s) and final extension for 5 minutes. In each run, negative and positive controls were included.
The PCR products amplification was analyzed by gel electrophoresis (1.5 Agarose in Tris-Acetate EDTA buffer) staining with ethidium bromide. The visualization was carried out using Gel Doc XR Imaging System (Bio-Rad).
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