26 Feb 2012

Purification of Plasmid DNA

Following the initial characterization, it's possible to purify additionally some or perhaps all the plasmid DNAs simply by RNase digestion and removal having normal solvents. This particular more purified DNA is suitable for strategies for example DNA sequencing, subcloning or even this production regarding gene probes. In order to purify plasmid DNA as soon as the isolation practice, any residual RNA as well as contaminating protein are usually removed. This specific purification action involves two major measures, that are, initial, removing residual RNA by making use of RNase in order to digest RNA and, second, extract contaminating proteins making use of organic solvents, phenol-chloroform.


a.    RNase A: Make up as being a solution inside water with 10 mg/mL, Temperature pertaining to 10 minutes in a boiling water bath or perhaps warming prohibit to eliminate just about any DNase activity. Aliquot and retail store at -20oC.

b.    4 M Ammonium acetate.

c.    Chloroform= A 24: 1 mix of chloroform and also isoamyl alcohol. Store at 4oC.

d.   Phenol/chloroform= 25: 24: 1 mix of TE-equilibrated phenol, chloroform, as well as isoamyl alcohol. Store with 4oC.

e.    100% Ethanol.

f.     Sterile wooden toothpicks.


a.    Add 50 microliters connected with 4 M ammonium acetate that contain 200 micrograms/mL RNase A for you to every single miniprep and also incubate it with room heat range intended for 20 min.

b.    Add 100 microliters regarding phenol/chloroform to every DNA preparation.

c.    Vortex briefly in addition to centrifuge at high speed for 2 minutes in a microfuge. Get rid of the top layer made up of your DNA along with place it within a new sterile tube.

d.   Add 100 microliters involving chloroform to be able to every tube.

e.    Vortex briefly in addition to centrifuge with high speed in a microfuge for 2 minutes. Eliminate the DNA in the top layer along with place it inside a second sterile tube.

f.     Regarding phenol/chloroform extractions avoid eliminating materials from the interface.

g.   Add 200 microliters associated with 100% ethanol for you to each tube.

h.   Shake briefly to be able to precipitate this DNA and centrifuge at large speed regarding 5 min on room temp.

see more: Biotechnology

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