LAMP was performed in a 0.2-ml microtube with a 20-μl reaction mixture containing 2 μl plasmid standard, 1.6 μM each of FIP and BIP, 0.2 μM each of F3 and B3, 0.8 μM LB, 1.4 mM each of dNTPs, 6.4 U Bst DNA polymerase, Large Fragment (New England BioLabs, Sumida, Tokyo, Japan), and 120 μM hydroxy naphthol blue trisodium salt (CAS No. 63451-35-4; Sigma–Aldrich Japan, Shinagawa, Tokyo, Japan) in LAMP buffer (20 mM Tris–HCl [pH 8.8], 8 mM MgSO4, 10 mM KCl, 10 mM (NH4)2SO4, 0.1% Tween-20, and 0.8 M betaine). The reaction mixture for RT-LAMP was identical to those described above for LAMP except that 5 μl RNA extract was used as template instead of 2 μl plasmid standard and 0.45 U cloned AMV Reverse Transcriptase (Life Technologies) was included in the reaction mixture. For the negative controls in RT-LAMP, the RNA extract was substituted with RNA extract from 1.3 × 107 TCID50/ml of feline calicivirus (FCV) or molecular grade water. The samples were placed in a thermocycler (Applied Biosystems 2720; Life Technologies) and incubated at 62 °C for 90 min. Successful gene amplification was indicated by a color change of the reaction solution from purple to sky blue under ambient light (Goto et al., 2009 (link)).
Applied biosystems 2720
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Applied Biosystems 2720 Thermal Cycler is a laboratory instrument used for the amplification of DNA samples through the polymerase chain reaction (PCR) process. It provides precise temperature control and cycling capabilities to facilitate the thermal cycling required for DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using applied biosystems 2720
1
Sensitive LAMP Detection of Viral RNA
2
Rice Genomic DNA Extraction and SSR Analysis
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The fresh leaves of rice were collected for genomic DNA extraction according to the cetyltrimethylammonium bromide (CTAB) method [21] , and the concentration of genomic DNA was determined by the ethidium bromide uorescent staining method [22] (link). Forty-three pairs of uorescent primers that can effectively distinguish the genetic differentiation of rice varieties were selected (Table S2). An Applied Biosystems 2720 thermal cycler (Applied Biosystems, USA) was used for PCR, and the reaction system volume was 10 µL, including 1 µL of DNA template (50 ng), 0.2 µL of each primer (forward and reverse primer), 0.8 µLdNTP mix (TAKARA), 0.05 µL Taq DNA polymerase (Biocolor BioScience, Shanghai), 2 µLPCR buffer (10 ×, including 2 mmol/L MgCl 2 ) and 6.75 µL double distilled water.The PCR conditions were as follows: 1 cycle at 94°C for 4 min; 30 cycles at 94°C for 40 s, 55°C for 30 s andb72°C for 30 s; 1 cycle at 72°C for 7 min. PCR products were separated and analyzed by a uorescent capillary electrophoresis system (ABI 3130xl Genotyper, Applied Biosystems, Genesky, Shanghai). The molecular weights were calculated by the biological software GeneMapper (version 3.7, Applied Biosystems, Inc., USA). SSR data reading was performed according to the size of ampli ed products, and the products with the same size were regarded as an allele.
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