RNA was extracted from synchronized parasite cultures at 20–24 h after percoll/sorbitol gradient centrifugation. RNA was extracted with the TRIZOL LS Reagent® as described (Kyes et al, 2000 ) and purified on PureLink column (Invitrogen) according to manufacturer's protocol. Isolated RNA was then treated with DNase I (TaKaRa) to degrade contaminating gDNA. cDNA synthesis was performed from 500 ng total RNA with PrimeScript™ RT Reagent Kit (TaKaRa) as described by the manufacturer.
Purelink column
Manufactured by Thermo Fisher Scientific
The PureLink column is a laboratory equipment designed for DNA and RNA purification. It provides a reliable and efficient method for the isolation and concentration of nucleic acids from various sample types. The core function of the PureLink column is to facilitate the extraction and purification of DNA or RNA, enabling researchers to obtain high-quality genetic material for downstream applications.
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3 protocols using purelink column
1
RNA Extraction and cDNA Synthesis
2
Genomic DNA and RNA Extraction for Malaria Parasites
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Genomic DNA was extracted as described (18 (link)). Briefly, iRBCs from a 20 ml cultures were lysed with saponin and the parasite pellet was washed with PBS and taken up in 200 μl TSE buffer (100 mM NaCl, 50 mM EDTA, 20 mM Tris, pH 8), 40μl of 10% SDS and 20 μl 6M NaClO4. This suspension was rocked for twelve hours and genomic DNA was extracted with phenol/chloroform. The resulting DNA was taken up in 100 μl dH2O. RNA was extracted from synchronized parasite cultures at 20–24 h after percoll/sorbitol gradient centrifugation (16 (link),17 (link)). RNA was extracted with the TRIZOL LS Reagent® as described (23 (link)) and purified on PureLink column (Invitrogen) according to manufacturer's protocol. Isolated RNA was then treated with DNase I (TaKaRa) to degrade contaminating gDNA. cDNA synthesis was performed from 500 ng total RNA with PrimeScript™ RT Reagent Kit (TaKaRa) as described by the manufacturer.
3
Yeast RNA Extraction and RNA-seq Library
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About 2–3 × 108 flash-frozen yeast cells were resuspended in Trizol (cell pellet:Trizol = 1:10) and vortexed for 15 s and incubated at 25°C for 5 min. Subsequently, 200 μl chloroform was added per 1 ml of Trizol–cell suspension and samples were vortexed for 15 s, incubated at RT for 5 min and centrifuged to recover the aqueous layer. The RNA in the aqueous layer were further purified and concentrated using PureLink Column (12183018A, Invitrogen). The RNA was eluted in 50 µl and store at −20°C if not used immediately. Store at −80°C for long term. Paired-end RNA-seq libraries were prepared using TruSeq stranded mRNA library preparation kit (20020594, Illumina).
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