DNA Purification / Plasmid MiniPrep

DNA Purification / Plasmid MiniPrep

Introduction of the topic:

Hello today we are going to present to you plasmids and their purification process. Firstable plasmids are small circular pieces of DNA that are different from a cell's chromosomal DNA. They are holders of genetic information that can be transmitted from one cell to another.

(They are not essential to cell survival, but they can provide one or more functional benefits to the host cell, for example: resistance to antibiotics.)

Lysis

1- We start by using a culture of bacteria that has been left to grow overnight.

 Wet aking 1,5ml of Escherichia coli culture and placing it inside a small test tube. Next, we have to centrifuge for 2 min,  in order to sediment the bacteria, so we nw have the bacteria at the bottom of the tub and at the top the supernatant . Then, drein the clarified the supernatant, leaving only with a concentrated cell solution

3 )After waiting for 1min, we add  250um of  lysis buffer (solution II) and mix gently by inverting the tube 4-6 times: this will break the cell walls and release their content into the test tube.

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5) Finaly we centrifuge for 5 min at maximom speed.

Now we have the plasmids that are released into the tube, but they are surrounded by many other cell components. So we are going to separate them from the other cell components.

Séparation

7-8)To get rid of the contaminants we discard the flow-through which doesn’t contain plasmid DNA. Then, we use another solution to wash our column and discard the flow-through, and after xe centrifuge for 2min. We must repeat this step twice to ensure maximum purification.

10)So we transfer the column to a clean test tube to avoid contamination.

Then, we add our last solution that is an elution buffer, and incubate at room temperature for two minutes. And last we centrifuge it for two minutes, so we have  the isolated plasmid

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