Isolation Of Plasmid Dna From A Bacterial Culture For Cloning

1554 Words Oct 15th, 2016 7 Pages
Objective:
We started by isolating a DNA plasmid from a bacterial culture for cloning. Then, we reverse transcribed mRNA to amplify it in a polymerase chain reaction. Finally, we amplified a specific gene (actin) through PCR.

Protocol:
A. Isolation of Plasmid DNA from a Bacterial Culture:

We were provided with a tube containing 1 ml of bacterial culture. We centrifuged it for 5 minutes at 10000 g. Then, we removed the supernatant by pouring it off and we used a small chem wipe to remove any excess media remained. Centrifuging will form a small pellet at the bottom of the tube. Using 250 l of cell resuspension solution, resuspend the cell pellet thoroughly by pipetting or vortexing. This will allow the cell lysis solution, which will be added next, to reach all cells. Then, add cell lysis solution to break the membranes, lyse cells, and release all the components of the cell in the tube. Invert the tube four time to mix all the components of the tube. However, do not shake vigorously or pipette or vortex the eppendorf tube because that may denature the DNA. After the cell is lysed, some proteins will be produced, which can have a negative impact of the quality of the isolated DNA. Therefore, you must add 10 l of alkaline protease solution to denature all the proteins. Invert the tube couple of times and incubate at room temperature. Make sure that you do not leave the alkaline protease solution in the tube for more than 5 minutes because it can start denaturing DNA. Then,…

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