Laurence Stuart Dawkins-Hall
Popular answer For 'clean sterile' preps like mammalian TC cells genomic DNA extraction columns work well. Keep in mind however that columns tend to have a saturation capacity (mini preps ~ 100ug) so make sure that for plasmid/Genomic minipreps you don't exceed > ~10^7 cells (~ T-75 flask) and therefore if necessary use midi columns for more starting material If you saturate your column because of loading on too much material the gDNA will stick to the matrix and you will fail to elute For tissue, Trizol extraction works effectively; and initial Trizol/chloroform extraction followed a column based supplementary clean up will produce the highest quality of genomic DNA of all An alternative for genomic DNA extraction is proteinase K digestion (37C over night or 55C for 1 hour) followed by either phenol/chloroform extraction and isopropanol precipitation and 70% ethanol wash of the resulting pellet or Trizol/chloroform extraction followed by isopropanol precipitation and 70% wash. In my experience and when done proficiently this produces similar yields and quality of final material to method 2 For plasmid DNA - you havn't specified what type of DNA you are attempting to purify - starting from bacterial cells either manual methods based on alkaline lysis followed by phenol chloroform/Trizol_Chlorform extraction; ethanol precipitation and 70% wash or alternatively kit based alkaline lysis of starting material followed by column based extraction produce similar quality of purified DNA although some commentators would suggest that column based methods produce superior DNA with proficient use In general terms Nibras all of these methods tend to work well when carried out properly and with some troubleshooting modifications incorporated PROVIDING you pay attention to the quality and quantity of starting material, especially for column based methods, where column overload can lead to your DNA becoming stuck in the column matrix with failure to elute/recover. Manual extraction methods are more forgiving in this sense
Hi Nibras
- In short yes; but DNA quality and recovery is also dependent on the quality and quantity of starting material. Generally speaking larger masses of tissue with 'extra impurities' like lipids are best extracted by manual based Trizol/phenol/Chloroform and/or proteinase K digestion in terms of good recovery and low RNA contamination and especially manual extraction followed by column based supplementary clean up in terms of good recovery; low RNA contamination and highest 260/280 (1.7) and 260/230 ratios (> 1.0). In contrast for smaller amounts of clean material, e.g. tissue culture cells, column based methods are very effective for genomic DNA. To elaborate
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Laurence Stuart Dawkins-Hall
For 'clean sterile' preps like mammalian TC cells genomic DNA extraction columns work well. Keep in mind however that columns tend to have a saturation capacity (mini preps ~ 100ug) so make sure that for plasmid/Genomic minipreps you don't exceed > ~10^7 cells (~ T-75 flask) and therefore if necessary use midi columns for more starting material If you saturate your column because of loading on too much material the gDNA will stick to the matrix and you will fail to elute For tissue, Trizol extraction works effectively; and initial Trizol/chloroform extraction followed a column based supplementary clean up will produce the highest quality of genomic DNA of all An alternative for genomic DNA extraction is proteinase K digestion (37C over night or 55C for 1 hour) followed by either phenol/chloroform extraction and isopropanol precipitation and 70% ethanol wash of the resulting pellet or Trizol/chloroform extraction followed by isopropanol precipitation and 70% wash. In my experience and when done proficiently this produces similar yields and quality of final material to method 2 For plasmid DNA - you havn't specified what type of DNA you are attempting to purify - starting from bacterial cells either manual methods based on alkaline lysis followed by phenol chloroform/Trizol_Chlorform extraction; ethanol precipitation and 70% wash or alternatively kit based alkaline lysis of starting material followed by column based extraction produce similar quality of purified DNA although some commentators would suggest that column based methods produce superior DNA with proficient use In general terms Nibras all of these methods tend to work well when carried out properly and with some troubleshooting modifications incorporated PROVIDING you pay attention to the quality and quantity of starting material, especially for column based methods, where column overload can lead to your DNA becoming stuck in the column matrix with failure to elute/recover. Manual extraction methods are more forgiving in this sense
Hi Nibras
- In short yes; but DNA quality and recovery is also dependent on the quality and quantity of starting material. Generally speaking larger masses of tissue with 'extra impurities' like lipids are best extracted by manual based Trizol/phenol/Chloroform and/or proteinase K digestion in terms of good recovery and low RNA contamination and especially manual extraction followed by column based supplementary clean up in terms of good recovery; low RNA contamination and highest 260/280 (1.7) and 260/230 ratios (> 1.0). In contrast for smaller amounts of clean material, e.g. tissue culture cells, column based methods are very effective for genomic DNA. To elaborate
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Seied Mehdi Miri
Dear Nibras
I agree with the valuable and comprehensive comments of Mr. Dawkins-Hall. The extraction and purification of high-quality DNA from some plant species is generally difficult due to the presence of polysaccharides, proteins, and DNA polymerase inhibitors such as tannins, alkaloids, and polyphenols. The presence of these compounds effects the quality and quantity of isolated DNA. Most of the DNA extraction methods are modified versions of cetyltrimethyl ammonium bromide (CTAB) extraction with some crop-to-crop limitations and differ in time and cost. The main cause of the differences in the CTAB protocol is the composition of cell walls and intracellular components such as nucleus mitochondria and cellulose. So a lot of research has been done to compare quality and quantity of DNA isolated using different extraction methods.
Abdel-Latif, A., & Osman, G. (2017). Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods, 13, 1. http://doi.org/10.1186/s13007-016-0152-4
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