Selecting a Genomic DNA Purification Method
The goal of genomic DNA isolation depends on what the applications of the DNA after isolation. Purity, source, quantity and quality of DNA are all issues that need to be addressed prior to genomic DNA extraction. A whole host of different methods, technologies and kits are available now to researchers to isolate genomic DNA from cells.
- Quantity of DNA needed
- Molecular weight and size of DNA
- Purity of DNA required
- Downstream applications of DNA
- Time available
- Ease of DNA extraction technique or method
- Expense or money available
Home-grown or laboratory specific DNA extraction methods often quite well for labs that have developed them and regularly use these protocols.
Note however these methods lack
standardization. Also the DNA yield and DNA quality are not always
reproducible from one person to the next.
Background on Genomic DNA Isolation and Purification
Generally, all methods involve the disruption and lysis of cells. This is followed sometimes by the removal of RNA (by RNAses, salt or other methods). Choosing which method to use will depend on many selection factors including:
DNA is isolated from proteins by several methods including digestion of proteins by the enzyme proteinase K. Proteins are removed subsequently by salting-out, organic extraction, or binding of the DNA to a solid-phase support (such as an anion-exchange column or silica technology).
DNA is finally recovered by ethanol precipitation or isopropanol precipitation.
In general, the separation of DNA from cells and cellular components can be divided into four stages:
- Cell disruption
- Lysis of Cell
- Removal of Proteins and Contaminants
- Recovery of DNA
In some genomic DNA isolation protocols, stages 1 and 2 are combined.
Materials Needed for Genomic DNA Extraction Method
Lysis Buffer for Genomic DNA Recipe:
50 mM Tris-HCl, pH 8.0
Genomic DNA Purification Method from Tissue Samples
- Select tissue sample to use. Make sure the sample is properly prepared and kept on ice. If the sample will not be used immediately for DNA extraction, then the samples must be stored properly in either -20°C freezer for longer term or 4°C for short term (few hours) usage.
- Cut tissue into smaller pieces. For liver or other soft tissue, don't worry about making fine pieces.
- Add tissue to a pre-cooled (dry ice) mortar, immediately add liquid nitrogen N2.
- Begin to grind tissue into fine powder. Add more Liq. N2 as needed. Transfer cold powder to 30 ml tube, leave uncapped so N2 can evaporate.
- Add 9 ml of per-warmed (5°C ) Lysis buffer and gently resuspend powder.
- Add 100 ul of 10mg/ml of Proteinase K, (i.e, 100 ug in solution). Incubate 55°C 1-18 hours. Mix gently periodically. Add 100 ul of Proteinase K after first 2 hours, Optimum: 3 hours adding Proteinase K at 1.5 hours.
- Treat sample very gently. Add 1 ml of 3M Na0Ac (pH 4.0), 10 ml of warm (55°C ) phenol, mix gently but throughly for 5-10 minutes.
- Centrifuge samples for 15 minutes.
- Repeat warm phenol extraction.
- Extract with equal volume (10ml) phenol: CIA (1 part phenol:1 part CIA: CIA= 23 parts chloroform:1 part isoamyl alcohol)
- Extract with equal volume (10ml) CIA extraction
- Upper phase should be relatively clear, If it contains large white clouts, or is very milky, (i) incubate at 68°C 15 min., and re-phenol extract, (ii) start again, but incubate longer with P-K.
- Transfer upper phase to new tube. Add 2 volumes of cold ethanol and mix gently (Salt: Na0Ac is already in solution). Using a hook and sealed pasteur pipet spool out DNA. Wipe off excess ethanol on the side of the tubes. Resuspend in 2-5 mls of TE. Make sure DNA is completely dissolved (leave on gentle shaker.)
- Add RNase mix (100 ug of RNase A, 10U of RNaseT1). Incubate 30 min 37°C. ** You could add the RNase before the proteinase K, incubate at 37°C for 1 hour and then start proteinase digestion. You could then skip step 15-.
- Add 100 ug of Proteinase K, incubae 37°C 30 minutes.
- Add 1/10 vol. 3M Na0Ac, Phenol extract once, Phenol:CIA extract twice, CIA extract once.
- Add 2.5 volumes of EtOH, put in -20°C 30 min. Centrifuge 20 min.
- Wash well with 70%, Remove all EtOH. If you dry, Do Not over DRY.
- Resuspend carefully, slowly in1-2mls TE (Tris-EDTA buffer). (1x or 0.1x).
Other DNA Protocols and Methods
Restriction Enzyme Digestion - all you need to know !
DNA Transformation - Contains History of DNA
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