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  Section: Biotechnology Methods » Molecular Biology
 
 
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Isolation of RNA from Free-Living Rhizobia

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Cells from 100 mL culture in early-log-phase GYPC were spun and resuspended in 1 mL sterile 25% sucrose. This solution was transferred to a sterile centrifuge tube and 6.5 mL M-STET, 0.5 mL lysozyme (10 mg/mL), and 0.4 mL 200 mM vanadyl ribonucleoides were added.

M-STET: 4% sucrose, 6% triton X100, 0.06 M Tris (pH 8), 0.06 M EDTA Incubate on ice for 5 min. Boil 2 min (or add SDS/sarkosyl, extract with phenol:chloroform, and precipitate), chill, and spin 10 min at 8000 rpm. To the supernatant, add 0.7 vol isopropanol, chill, spin again, and resuspend pellet in 5 mL.

5X Running buffer for RNA (formaldehyde) gels: (for 500 mL) 2 mL 0.5 M EDTA, 6.8 g Na acetate, 12.8 g MOPS (free acid), 9.0 g MOPS (base).
  1. Induce cells normally –10 mL of 0.2 OD600.
    Wash cells (grown overnight in ORS minimal plus yeast at 30° or 37°C) 2 times with nif media and diluting to 0.2 OD (10 mL each). Check 1 bottle for induction by acetylene reduction.
  2. After induction, add 0.5 mL vanadyl ribonucleosides directly through the septum into a vial with a syringe (vanadyl protects cells against oxygen).
  3. Put cells in plastic tubes and spin for 2 min at 8000 rpm in Sorvall.
  4. Resuspend in 10 M urea lysis buffer with a plastic pasteur pipette.
  5. Add 0.5–0.75 mL of phenol:chloroform (50:50) (“phenol” is phenol: m-cresol:hydroxyquinoline) to the microfuge tube.
  6. Vortex, place at 65°C for 2–3 min. Repeat. Vortex and spin 2–3 min in microfuge.
  7. Re-extract the liquid phase 2 more times at room temperature with phenol:chloroform. Avoid proteinaceous interphase when removing the upper liquid phase.
  8. Extract with chloroform 1 time.
  9. Precipitate with 100% ethanol, 0.3 M Na acetate (5.2) at –20°C overnight.
  10. Collect precipitate, wash 1 time with 70% ethanol, 1 time with 100% ethanol, and resuspend in 10 mM Na acetate (5.2). Treat for gels.

 
     
 
 
     




     
 
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