RNA Extraction Method for Cotton


Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  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
  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
  1. Collect young expanding leaves (or other tissue) and freeze in liquid N2 and store at –80°C.
  2. Pulverize tissue to a fine powder in a precooled mortar and transfer to a glass homogenizer.
  3. Heat RNA extraction buffer to 80°C and add to frozen ground tissue at a ratio of 5:1 (buffer:tissue) and homogenize for 2 minutes.
  4. Transfer homogenate to a 50-mL Oak Ridge tube containing 0.5 mg proteinase K per mL of extraction buffer and incubate with mild agitation on a rotary shaker at 100 rpm for 1.5 hr at 42°C.
  5. Add KCl to 160 mM and chill on ice for 1 hour.
  6. Centrifuge for 20 min at 12000 g.
  7. Filter supernatant through Miracloth and precipitate the RNA overnight in 2M LiCl at 4°C.
  8. Centrifuge for 20 min at 12000 g to collect RNA pellet. Wash pellet 2–3 times with 5 mL cold 2 M LiCl until supernatant is relatively colorless.
  9. Resuspend RNA pellet in 2 mL 10 mM Tris-HCl (pH 7.5) and clarify by centrifugation for 10 min.
  10. Add potassium acetate (KAc, pH 5.5) to 200 mM in RNA suspension and incubate for 15 min on ice.
  11. Remove salt-insoluble material by centrifugation.
  12. Precipitate RNA overnight by adding 2.5 volumes cold 100% ethanol and incubating at –20°C.
  13. Pellet RNA, wash with 70% ethanol, dry briefly under the vacuum, and suspend pellet in DEPC-treated deionized water of TE buffer.
  14. Determine yield by observing absorbance spectra between 220 and 320 nm. Yield should be about 800–1200 µg/g (RNA/fresh tissue).

RNA Extraction Buffer
  • 200 mM sodium borate decahydrate (Borax) pH 9.0
  • 30 mM ethylene glycol bis(β-aminoethyl ether)-N,N´-tetraacetic acid (EGTA)
  • 10 mM dithiothreitol (DTT)
  • 2% polyvinylpyrolidone, Mr 4000 (w/v) (PVP)
  • 1% sodium dodecyl sulfate (w/v) (SDS)
  • 1% sodium deoxycholate (w/v)
  • 0.5% Nonidet NP-40 (v/v) (NP-40)

Methods for Plant RNA Isolation

AMES/Chloroform extraction in our lab to extract total RNA from potato leaves for viroid analysis. The protocol is as follows:
- AMES Buffer (for 200 mL): 11.7g NaCl, 160 mL dH2O, adjust pH to 6.0, 0.40 g MgCl2, 8.21 g Na-acetate, 40 mL EtOH, 6.0 g SDS.
- Homogenize the plant material (usually leaves) and dilute the sap 1:5 in AMES. Then incubate for 30 min at 37°C followed by the addition of an equal volume of chloroform. Vortex and store on ice. Centrifuge the samples at 3000 rpm for 10 min. Total RNA is contained in the upper aqueous layer.