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Purification of plasmid DNA

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Purification of plasmid DNA

Postby chemistry_freako » Wed Jun 08, 2005 3:37 pm

In the purification of plasmid DNA (of E.coli), 6 reagents were used:
Cell resuspension solution, cell lysis solution, alkaline protease, neutralising solution, column wash solution, as well as nuclease free water.

Components of the reagents are as such:
Cell resuspension solution:
Tris-HCl
EDTA
RNase A

Cell lysis solution:
NaOH
SDS

Alkaline protease
Protease enzyme subtilisin (produced by Bacillus licheniformis)

Neutralising solution:
Potassium acetate
Tris- HCl
EDTA

Column wash solution:
potassium acetate
Tris- HCl
EDTA

So what exactly is the function of all the various components, of the various reagents, in obtaining a purified sample of the plasmid DNA puc19 from E.coli?
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Postby DevGrp » Thu Jun 09, 2005 9:09 am

this site should help

http://lsvl.la.asu.edu/resources/mamaji ... aline.html


Never used Protease myself, and not sure why you have a "Column wash solution" step


And generally Tris is a pH buffer and EDTA celates metal ions which inhibits nucleases.
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Oh..i see

Postby chemistry_freako » Sat Jun 11, 2005 3:01 pm

i see...thanks!
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