Answer to Question #128200 in Biochemistry for Benjamin Afful

Question #128200

a.      During the isolation of TOL plasmid from Pseudomonas putida, it was observed that the concentration of the plasmid DNA per 50 mL of sample was extremely low. Describe a technique you could employ to increase the concentration of the TOL plasmid.

Expert's answer

Molecular biology is the study of biological macromolecules that are involved in the various biochemical processes in the living organism. To increase the concentration of the TOL plasmid, use a bigger prep kit and a large volume initial culture. And if the quantity of the plasmid is low, likewise expect a low yield. Therefore, it is recommendable to use a high yield of plasmid. While undertaking the experiment, keep on checking on the troubleshooting section to ensure that the results is as per the requirements to avoid some weird outcomes. To ensure that the bacterial cultures are properly inclined, give a guarantee by maintaining the shaking at a constant of 200RPM (Ideal 220 to 230) for a maximum growth of bugs.

Indeed, if you're performing a midi-maxi prep you'll have a pellet after the isopropylic alcohol precipitation and ethanol washes, you can cut a p1000 tips with enough width to ensure you can aspirate the whole pellet and then you can aspirate all the pellets and place all of them into a single 2mL vial. You can then centrifuge again and air dry the "megapellet" and resuspend it into desired volume of nuclease free water.

if you're performing minipreps with an in-colums final DNA eluition step, you can place all the eluition products of multiple preps into a single 2 mL vial and precipitate the plasmids by adding 1/10 sodium acetate 3.5 M pH 5.4 (also 3.0 M PH 5.5 works fine) and 2 volumes of absolute ethanol. Wash the pellet with 70% ethanol, air dry the pellet and again you can resuspend into the proper volume of nuclease free water.

If you want also to rise your plasmid purity you can also perform a phenol-chloroform extraction instead of Na-Ac/ethanol precipitation. In this way you'll have an increased amount of plasmid into desired volume of solution.

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06.08.20, 16:02

Thank you

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