Parasite lines were synchronized with sorbitol for two life cycles (13 (link)). 2-ml cultures were seeded with a mixture of two parasite lines with 1% total parasitemia in 4% hematocrit. Cultures were grown at 37 °C with 40 rpm shaking. 90% of the cultures was taken every generation to isolate genomic DNA. The genomic DNA was analyzed by allelic discrimination quantitative PCR with the primers and probes listed in Table 1 ; the polymorphism site is underlined (Invitrogen). Two biological replicates, each done in triplicate, were carried out for six generations. Allelic discrimination assays were performed with TaqMan Universal PCR Master Mix with no AmpErase UNG (Applied Biosystems) on a 7900 HT real time PCR machine (Applied Biosystems). PCR cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 56 °C for 60 s.
Taqman universal pcr master mix with no amperase ung
Manufactured by Thermo Fisher Scientific
Sourced in United States
TaqMan Universal PCR Master Mix with no AmpErase UNG is a ready-to-use solution designed for real-time PCR applications. It contains all the necessary components, including a DNA polymerase, dNTPs, and buffer, to perform PCR reactions. The mix does not contain the UNG (Uracil-N-Glycosylase) enzyme, which is often used to prevent carry-over contamination.
Automatically generated - may contain errors
Lab products found in correlation
7900ht real time pcr machine, by Thermo Fisher Scientific (1 mentions)
Reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Applied biosystems 7900ht system, by Thermo Fisher Scientific (1 mentions)
Rq manager software v1, by Thermo Fisher Scientific (1 mentions)
Mirvana isolation kit, by Thermo Fisher Scientific (1 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using taqman universal pcr master mix with no amperase ung
1
Quantitative Allelic Discrimination of Synchronized Parasites
2
Quantifying miRNA Expression via RT-qPCR
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The miR reverse transcription was conducted using a Reverse Transcription Kit (Life Technologies, Carlsbad, CA, USA). 0.5 μM miRNA PCR primers (RiboBio, Guangzhou, China) were used for PCR amplification. Real-time PCR was performed with TaqMan Universal PCR Master Mix with No AmpErase UNG (Applied Biosystems, Life Technologies, USA) on an Applied Biosystems 7900HT System (Applied Biosystems, Life Technologies, USA). Raw Ct values were calculated using the RQ Manager software v1.2 (Applied Biosystems, Life Technologies, USA) with automatic baseline and threshold settings. The miRs were normalized to the level of U6 snRNA.
3
Quantitative Analysis of miRNA-210 Expression
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Total RNA was extracted from cells with the mirVana Isolation kit (Applied Biosystems). cDNA was synthesized from total RNA using reverse transcription primers that were specific to miR-210 and the Taqman miRNA reverse transcription kit (Applied Biosystems). To assess miRNA, the cDNAs prepared from the specific reverse transcription reactions were used in PCRs containing Taqman Universal PCR Master Mix with no AmpErase UNG and premixed Taqman assays (Applied Biosystems). The Taqman assays for miRs only detect the mature miRs. Reactions were carried out in a 96-well optical reaction plate with optical caps (Agilent Technologies) in a Mx3000p Real-Time PCR Detection system spectrofluorometric thermal cycler (Agilent Technologies). Reactions proceeded with an initial 10-min incubation at 95°C, followed by 40 cycles of amplification: 95°C for 15 s and 60°C for 1 min. Fluorescence was measured in real time; the cycle threshold (Ct) values were calculated using the Mx3000p algorithm (Agilent Technologies). Comparative quantitation was performed by comparing the Ct value obtained from the amplification of a given target miRNA with that determined for the normalizer RNU6B. Relative miRNA abundance was calculated using the –ΔΔCt method. Student’s t test was used for statistical analysis.
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