A liquid stationary S. Typhimurium culture grown in Luria broth, Miler formulation (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl) was pelleted and lysed overnight at 37°C in lysis buffer (20 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.15 mg/mL proteinase K and 0.2% SDS). The bacterial lysate was separated using a phase lock gel (phase lock gel heavy, 5PRIME, Quantabio) and Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) extraction. The DNA was precipitated from the aqueous phase using a final concentration of 50% isopropanol and 0.3 M NaCl and the pellet was washed in 70% ethanol and resuspended in 10 mM Tris-HCl pH 8 and 1 mM EDTA.
5prime
Manufactured by Quantabio
5PRIME is a versatile laboratory equipment designed for nucleic acid analysis. It is a high-performance device that enables precise manipulation and processing of DNA, RNA, and other genetic materials. The core function of 5PRIME is to facilitate various molecular biology techniques, such as PCR amplification, nucleic acid purification, and sample preparation. The product specifications and capabilities are tailored to support a wide range of research and diagnostic applications in the life sciences industry.
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5 protocols using 5prime
1
Genomic DNA Extraction from S. Typhimurium
2
Yeast RNA Extraction and Purification
3
Yeast RNA Extraction and Purification
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RNA was extracted from 25 OD of yeast corresponding to each sample by
modification of a previously described method55 . To each sample, 0.5 g of 0.5 mm acid
washed beads (Sigma-Aldrich), 0.5 mL of RNA ISO buffer (500 mM NaCl, 200 mM
Tris-Cl pH 7.5, 10 mM EDTA, 1 % SDS) and 0.5 mL of phenol-chloroform-isoamyl
alcohol pH 6.7 (PCA, Fisher Scientific) was added. Samples were lysed with 10
cycles that each consisted of vortexing for 20 seconds and incubation on ice for
30 seconds. 1.5 volumes (relative to starting amount of RNA ISO Buffer) of RNA
ISO Buffer and of PCA were added, and samples were centrifuged at 4°C to
separate phases. The aqueous layer was transferred to a pre-spun phase-lock gel
(heavy) tube (5PRIME/Quantabio); an equal volume of PCA was added and mixed
prior to centrifugation at room temperature to separate phases. The aqueous
layer was transferred to 2 new tubes for ethanol precipitation with 2 volumes of
100% ethanol followed incubation at −80°C for 1 hour to overnight.
Precipitated RNA was pelleted by centrifugation at 4°C. Each pellet was
dissolved in 25 μL nuclease-free water and combined into 1 tube per
sample. Co-purifying DNA was digested with 20 U of Turbo DNase (Invitrogen) at
37°C for 4 hours. RNA was cleaned up with the GeneJET RNA Purification
Kit (Thermo Fisher Scientific) and eluted with 50 μL of DEPC-treated
water.
modification of a previously described method55 . To each sample, 0.5 g of 0.5 mm acid
washed beads (Sigma-Aldrich), 0.5 mL of RNA ISO buffer (500 mM NaCl, 200 mM
Tris-Cl pH 7.5, 10 mM EDTA, 1 % SDS) and 0.5 mL of phenol-chloroform-isoamyl
alcohol pH 6.7 (PCA, Fisher Scientific) was added. Samples were lysed with 10
cycles that each consisted of vortexing for 20 seconds and incubation on ice for
30 seconds. 1.5 volumes (relative to starting amount of RNA ISO Buffer) of RNA
ISO Buffer and of PCA were added, and samples were centrifuged at 4°C to
separate phases. The aqueous layer was transferred to a pre-spun phase-lock gel
(heavy) tube (5PRIME/Quantabio); an equal volume of PCA was added and mixed
prior to centrifugation at room temperature to separate phases. The aqueous
layer was transferred to 2 new tubes for ethanol precipitation with 2 volumes of
100% ethanol followed incubation at −80°C for 1 hour to overnight.
Precipitated RNA was pelleted by centrifugation at 4°C. Each pellet was
dissolved in 25 μL nuclease-free water and combined into 1 tube per
sample. Co-purifying DNA was digested with 20 U of Turbo DNase (Invitrogen) at
37°C for 4 hours. RNA was cleaned up with the GeneJET RNA Purification
Kit (Thermo Fisher Scientific) and eluted with 50 μL of DEPC-treated
water.
4
Phenol-Chloroform DNA Extraction Protocol
5
Chromatin Compaction Analysis of LNCaP Cells
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One million LNCaP cells were plated for 3 days and half of media was replaced to fresh media when cells were treated with DMSO or 20µM OSMI-4 for 24 hours. Cells were fixed by adding formaldehyde in 1% final concentration for 10 minutes at room temperature and fixation was stopped by addition of 125mM glycine (final) for 5 minutes at room temperature. Plates were washed with ice cold PBS, cells pelleted by centrifugation (1000rpm, 10 minutes) and lysed in 0.5% SDS, 0.5% TritonX 100, 150mM NaCl, 10mM EDTA, 50mM Tris-HCl pH8.0 containing 1X protease inhibitor. Samples were sonicated using Bioruptor. Input sample was taken and the rest of the sample was subjected to three subsequent phenol:chloroform:isoamyl alcohol (25:24:1) extractions to isolate protein-free DNA in the aqueous phase using columns (#2302830, 5Prime, QuantaBio). All the samples (including input) were treated with proteinase K for 1 hour at +55˚C, DNA de-crosslinked at +65˚C for 4 hours and purified using standard phenol-chloroform extraction. qPCR was used to evaluate chromatin compaction relative to input.
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