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  Section: Biotechnology Methods » Molecular Biology
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Methylene Blue DNA Staining

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. Load 2–5X the amount of DNA that would give bands of moderate intensity on an ethidium bromide-stained gel. Typically, this is something on the order of 0.5–2.5 µg of a 1-kb fragment on a 30 mL 1% mini gel. These numbers are guesstimates so your mileage may vary.
  2. Run the gel normally and then place it in a 0.002% methylene blue (w/v, Sigma M-4159) solution in 0.1X TAE (0.004M Tris 0.0001 M EDTA) for 1–4 hrs at room temperature (22°C) or overnight at 4°C. Diffusion of the DNA does not seem to be a problem for fragments as small as 100 bp (3% Nusieve:1% agarose gel). This avoids background issues associated with staining with 0.02% methylene blue for 30–60 min and then destaining for a long time.
  3. If destaining is needed to increase the visibility of the bands, place the gel in 0.1X TAE with gentle agitation, changing the buffer every 30–60 min until you are satisfied with the degree of destaining.

This method primarily eliminates the damage of DNA by UV irradiation. DNA isolated from MB stained gels should transform frozen competant E. coli (XL1- Blue and DH5) cells on the order of 20–50-fold more efficiently than EB-isolated DNA. Factors influencing improved efficiency are: time factor (degradation, etc.), transilluminator wavelength and intensity, and the %AT of your DNA to mention a few. One of the advantages of MB staining is the elimination of several of the variables.

Both FMC GTG agarose and Nusieve GTG perform very well. Synergel is incompatible with MB (very high background). MB should be compatible with polyacrylamide (even less of a background problem).

Nuieve:Agarose (3:1, 4% final) gels stain very nicely and dsDNA as small as 75 bp is easily visualized.

Recovery of DNA from Low-melting-temperature Agarose Gels
Ultraviolet radiation and EtBr in a gel are dangerous. Wear protective goggles and gloves to protect the eyes and EtBr contamination.

  1. Digest the plasmid-DNA (up to 20 µg)-containing insert.
  2. Pour a gel containing the appropriate concentration of low-meltingtemperature agarose.
  3. Mix the samples of DNA with tracking dye, heat shock at 65°C for 5 min, transfer in ice, and load onto the gel.
  4. Carry out electrophoresis at 12 volts overnight. DNA of a given size runs slightly faster through gels cast with lowmelting- temperature agarose than through conventional gels.
  5. Take a picture.
  6. Using a handheld UV light with a long wavelength (to minimize the damage to the DNA), cut out insert bands using a scalpel. Cut the gel as close to band of interest as possible and transfer it to a clean 1.5 mL MFT.
    Check or draw the removing band on the picture for a record of which band was eluted.
  7. Add ≈ 5 volumes of H2O to the slice of agarose.
  8. Melt the agarose at 65°C for 1 to 5 min. Vortex for 20 sec and store at –20°C. Further Separate the DNA from the Agarose.
  9. Spin the tubes (4K, 10 min, 20°C).
  10. Transfer the supernatant with a micropipette (P-20) into new MFT. The white substance at the interphase is powdered agarose.
  11. Re-extract the agarose phase once with phenol:chloroform and once with chloroform.
  12. Transfer the aqueous phase to a MFT, bring up to 105 mL with H2O, add 35 µL NH4Ac and 1050 µL EtOH, and invert to mix.
  13. Centrifuge 5 K, 20 min at 4°C.
  14. Take supernatant, rains with 70% EtOH, dry and resuspended in 5–10 µL TE. And store at –20°C.


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