Plasmid DNA Isolation – Improved Yield, No Kit

This improved plasmid DNA isolation has greater yield than most any column based method and costs about 5 cents. A number of steps have been optimized to produce large quantities of ultra pure DNA. Yield can exceed 40 micrograms plasmid per 1.7 mL culture. The low cost makes this procedure friendly to any lab on a budget or labs that wish to use money on more pressing commodities, like data. A longer version of this video with more tips can be found here.

Short version of Plasmid DNA Isolation (Recommended).

Download a print Plasmid DNA Isolation Protocol here.

Extracción de ADN plasmídico-Alto rendimiento, económico

Procedimiento de extracción de ADN plasmídico de alto rendimiento y económico, basado en un método mejorado de lisis alcalina. Este protocolo puede producir 43 microgramos de ADN de alta calidad a partir de 1.7 mL de cultivo bacteriano. Además es más rápido y cuesta solo 5 centavos. No requiere kits caros, lo que es perfecto para laboratorios con poco presupuesto.

Puedes descargar el protocolo aquí Extracción-de-ADN-plasmídico.

2 thoughts on “Plasmid DNA Isolation – Improved Yield, No Kit”

  1. What causes alkaline lysis for plasmid isolation to produce only short dna fragments on gel?

    I am following alkaline lysis protocol very carefully, with special attention to the correct pH of all the three solutions – TE with glucose, NaOH with sds and K-acetate with glacial acetic acid pH 5.2, etc. On my gels after restriction linearization (carefully chosen no to cut inside the gene) I get only small fragments that are somewhat smeared. I also did a phenol:chloroform extraction to clean up the DNA before digestion and remove any residual protein. Are there some tips to getting this right? I let the NaOH/sds with cell pellet rest on ice for 2 minutes before using the potassium acetate solution, and I did not vortex at all. Is that 2 minutes too long? Some protocols do not recommend this 2-5 minute incubation.
    thank you! – Amy J.

    1. Amy, I do not think I have enough information to help you with certainty. I don’t know if there is a way you can share a picture of your gel. I suggest you use the downloadable protocol on this page exactly as it states and use my video to help visualize the steps. The neutralization solution (with acetic acid and potassium acetate) should be added immediately after the lysis solution is added and mixed gently. Do not wait for 2 minutes. Also, try using controls like running uncut DNA along with your restriction digest.

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