This week we continue with extracting plasmids transformed into bacteria that have grown in a liquid culture, using a commercially available kit.
Our procedure started with centrifuging the liquid cultures to pellet the bacterial cells. Then, the cells were lysed (broken apart) using an alkaline buffer so that we could obtain the plasmids in the cells, and the lysates were cleared by centrifugation.
We then add the supernatant to the extraction column, which contains a silica membrane. This allows the plasmid to adsorb in a high-salt environment: the negative-charged silica interacts with the positively-charged sodium ions, which in turn interacts with the negatively-charged plasmids. To elute the plasmids, we apply a low-salt buffer to replace the sodium ions, and as a result, the plasmids are washed out of the extraction column into our collection tube.
A few of us stayed on for the optional restriction digest session. Here, we showed a way to check that we’ve purified the correct plasmid by using restriction enzymes, which cut our plasmids at specific DNA sequences on the plasmid. Different plasmids with different sequences will produce different DNA fragment sizes when digested by the same set of restriction enzymes. Today we used the XbaI and SpeI restriction enzymes. We then showed this in gel electrophoresis, in which the DNA fragments are separated in our 0.8% agarose gel as they migrate along the electric field applied with different speeds according to their sizes.
We were able to see bands corresponding to 2,800 base pairs with the purified plasmids, but the resolution of the gel wasn’t high enough to resolve restriction digest products – this could be improved by increasing the voltage or the run time for the gel. Tune in next week for our final workshop, when we test our genetic circuit!