Purification DNA

Proteases [DNase free ] are added to the DNA solution to decrease the amount of protein that can remain associated. After the protease action, the entire procedure of DNA extraction is repeated, and the DNA dissolved again in TE buffer and dialyzed against TE buffer to eliminate any proteases and peptides. Professors and students can also refer to the following website for DNA extraction and purification http //gmotraining.jrc.it/docs/ Session04.pdf 

Resuspend pellet in 5 ml of pre 2-BE buffer (25 mM boric acid/Tris (pH 7.6) containing 1.25 mM Na2EDTA and 100 mM NaCl). 

Wash pellet with absolute EtOH, vacuum dry pellet, resuspend in TE buffer. 

20 g CTAB = Hexadecyl trimethyl ammonium bromide 200 ml dH20 

Dissolve CTAB in H2O first, add Tris, then EDTA, stir overnight, then add NaCL 

The extracted DNA is dissolved in 400 il TE buffer, letting it sit at room temperature for at least 30 min. Then tap the tube gently to disperse precipitate. If it does not dissolve, incubate it at 60 °C, but for no more than 10 min. Transfer to Eppendorf tube. The dissolved DNA samples can be stored for short term at 4 °C, for longer periods, however, — 20°C is necessary. 

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G-25 M PP PE 1.5 9 28 Open Leur Taper Gravity Flow 0.15% Kathon CG DNA purification (>10 mers) 0.5-ml sample... 

Fig. 7. Unwrapped P-CAC cylinder showing the configuration for the Plasmid DNA purification...

Wizard SV 96 Plasmid DNA Purification System (Promega Corp., Madison, Wl, USA). 

For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to plasmid preparation using the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of resultant plasmid solution is applied to agarose gel electrophoresis to examine the yields of plasmids. 

Three to several colonies for each sample are inoculated into the same 600 pL of LB medium containing 50 pg/mL of kanamycin and cultured at 37°C overnight. For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to the plasmid preparation with the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of the resultant plasmid solution is directly applied to agarose gel electrophoresis for estimation of the size of plasmid in a form of covalently closed circular. Similarly, another 1 pL of the plasmid solution is treated with S fi/Pmel and analyzed by agarose gel electrophoresis for an insert size check. This plasmid solution set is the final product and is reserved in a freezer. 

Two microliters of pFlK-ORF plasmid solutions prepared with the Wizard SV 96 Plasmid DNA Purification System are treated with 5 U of S ifi and 5 U of Pmel in a 10 pL reaction volume containing lx Flexi Digest Buffer at 37°C for 60 min, and then the enzymes are inactivated by incubating at 65°C for 20 min. 

Clones with ampicillin resistance but without kanamycin resistance are examined with the CloneChecker System. By this antibiotic selection, the undesired vector heterodimer is removed. If both of the clones are false, additional clones are analyzed using the same protocol. When appropriate clones are obtained for almost all of the genes, they are inoculated from the ampicillin plates to prepare plasmids in a 96-well format by using the Wizard SV 96 Plasmid DNA Purification System see Note 6). 

The plasmid preparation using the Wizard SV 96 Plasmid DNA Purification System follows the protocol from Promega, except that TE buffer instead of H O is used for the final plasmid elution buffer. This alteration is applied to all the plasmid preparation processes in this chapter. 

Repeat the phenol/chloroform/isoamylalcohol extraction step (Section 3.3.3 steps 3-5). Alternatively, use a DNA purification kit, which also provides good results. 

Electrophoresis apparatus (1.5 mm x 16 cm x 14 cm) Silica DNA purification column (QIAquick, Qiagen)... 

There are different DNA purification protocols for different bacterial organisms, based on their different cell wall compositions. Purification of macromolecular DNA can be achieved through subsequent steps of phenol extraction (using Tris-saturated phenol to prevent the DNA from getting redissolved in the phenol). The... 

DNA purification Solvent extraction and precipitation, gel electrophoresis, chromatography size exclusion, ion exchange, solid-phase extraction, SPRI, affinity purification... 

An automated DNA purification system called a PlateTrak system [30] based on SPRI protocol has been developed by PerkinElmer Life Sciences, Inc., in collaboration with the Center for Genome Research at the White-... 

CRS Robotics Corp. (Burlington, Ontario, Canada) CRS DNA purification system SPRI... 

M. Stevens and K. McKeman, Automation of DNA Purification Using the PlateTrak Automated Microplate Processing System, Application note AN004-CCS, Packard Bioscience Co., Meriden, CT, Nov. 2000. 

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