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  Section: Plant Lab Protocols
 
 
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Methodology for Nucleic Acids

 
     
 
Rapid Isolation of Plasmid DNA (Miniscreen)
 
Procedures for a large-scale isolation arid purification of plasmid DNA are elaborate, time-consuming and require ultracentrifugation. However, there shall be occasions when one has to analyze a large number of samples for the presence of a plasmid and also do not need either to isolate large quantity of plasmid DNA or of high purity. There are several procedures that describe the isolation of small amounts of partially purified plasmid DNA from a large number of bacterial clones in a relatively short period of time. One such 'miniscreen' procedure is described below. This method does not require a large input of cells and can be carried out in small Eppendorf tubes.
 
 



Principle

The isolation procedure is based upon the release of soluble high MW DNA from disrupted cell wall and membranes, dissociation of nucleoprotein complexes by denaturation and proteolysis and the separation of DNA from other macromolecules.
 
 



Materials

Solution A (pH 8.0)
      25mM Tris-HCl                              30.3mg
      50mM glucose                               90mg
      Lysozyme                                       100mg (freshly added before use)
      Water                                               10mL
Solution B
      0.2M NaOH                                     0.8g
      1.0% SDS                                       1.0g
      Water                                               100mL
Solution C
      3M sodium acetate (pH 4.8)        24.6g
      Water                                               100mL
   Adjust pH with glacial acetic acid and then make up the volume
Solution D
50mM Tris-HCl (pH 8)                  0.60g
100mM sodium acetate               8.20g

      Water                                               100mL
 
 
Procedure
1.
Transfer 1mL of an overnight cell culture (E.coli JA 221 carrying plasmid pBR 328 or any other strain) into an Eppendorf tube (See 'Isolation of plasmid DNA'). Sediment the cells by centrifuging briefly in the microfuge. Drain off excess liquid.
2.
Resuspend the cells in 100mL of solution A. Incubate on ice for 30 min.
3.
Add 200mL of freshly prepared solution B. Vortex briefly and keep on ice for 5 min.   The cells will lyse immediately and the solution will become viscous.
4.
Add 150mL of solution C. Vortex briefly and keep on ice for 60 min. The bulk of chromosomal DNA and cell material will precipitate into a white viscous clump. Remove this by centrifuging in the microfuge for 5 min.
5.
Transfer 500mL of the cleared lysate to a clean Eppendorf tube. Add 1mL of ethanol, mix thoroughly, and cool in a freezer (-70°C) to precipitate DNA. Sediment the DNA by spinning for 5 min.
6.
Discard the supernatant from the DNA pellet and dissolve the precipitate in 100mL of solution D. Add two volumes of ethanol, mix and store in the freezer for 10 min. Pellet the DNA by centrifuging for 2 min.
7.
Repeat step 6 twice more. Remove residual ethanol from the final DNA pellet by drying under vacuum. Dissolve the DNA in a suitable buffer for further analysis. Test the plasmid DNA preparation by agarose electrophoresis.
 
 
References
1.  Clewell, D B and Helinski, D R (1972) J. Bacteriol 110 1135.
2.  Marko, M A, Chipperfield, R and Birnboim, H C (1982) Anal Biochem 121 382.
 
     
 
 
     




     
 
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