Difference between revisions of "One step 'miniprep' method for the isolation of plasmid DNA"
Revision as of 04:38, 24 April 2006
All 'miniprep' methods reported so far for the isolation of plasmid DNA involve multiple pipetting, extraction, centrifugation and changes of minifuge tubes. For screening large number of samples, they are therefore cumbersome, time consuming and not economical.
The technical report below by Chowdhury, K. (1991) is a very fast, simple and one step 'miniprep' procedure. The quality and quantity of DNA obtained by using this procedure is similar to those obtained by the other commonly used procedures of Serghini et al. (1) or Birboim and Doly (2). According to this procedure, the bacterial culture is directly extracted with a mixture of phenol-chloroform-isoamylalcohol and the liberated DNA is precipitated with isopropanol. This method is now being used routinely in our laboratory for isolating plasmids upto 12kb in size. A detailed description of the method is presented below:
1. Take 0.5ml of overnight E.coli culture in a microfuge tube. We routinely grow our cells in 'standard 1' bacteriological media supplied by Merck, Germany. 2. Add 0.5ml of phenol:chloroform:isoamylalcohol (25:24:1). The phenol was saturated with TE (10mM Tris, 7.5, 1mM EDTA) prior to mixing with chloroform and isoamylalcohol. 3. Mix by vortexing at the maximum speed for 1 minute. Alternatively, vortex for 10 seconds and then transfer to eppendorf mixer or an over-the-top rotator for 5 minutes. 4. Spin at 12,000g for 5 minutes. During the spin, prepare microfuge tubes with 0.5ml of isopropanol. After the spin, remove carefully about 0.45ml of the upper aqueous phase leaving the interphase undisturbed and add it to the isopropanol. Mix well and spin immediately at 12,000 g for 5 minutes. Addition of salt and cooling is unnecessary. 5. Pour off the supernatant, add carefully 0.5ml of 70% ethanol to the side of the tube, pour off. Repeat the washing once more. Vacuum dry the pellet and suspend in 100ul/ml RNAse). About 5-10ul of this DNA can now be cleaved with appropriate restriction enzyme(s) for analysis.
- Chowdhury, K. (1991) One step 'miniprep' method for the isolation of plasmid DNA. Nucl. Acids Res 19:10 2792
- Serghini, M.A. Ritzenthaler, C., and Pinck, J. (1989) Nucl. Acids Res 17, 3604
- Birnboim, H.C., and Doly, J. (1979) Nucl. Acids Res. 13, 1513 - 1523.
- Sterile LB broth works very well in this protocol
- In step 1, one can pipette 1.5ml of broth spin the microfuge tube, decant 1ml and leave behind 500ul to resuspend the pellet and continue as from step 2. This maximizes the total yield of plasmid.
- The largest band appearing after running a gel is usually genomic DNA. I run appropriate control markers.