Introductory Lab Session: 10th July 2009
Experiment Recap and Introduction
In the previous experiment, we had managed to prove that most of the cultures of E.coli which had passed negative selection did include plasmid DNA. The only E.coli cells not to contain any plasmid were the colony 9 cells but that could be down to several reasons; not all of them down to biology.
The next step is to show that these plasmids contain the BioBricks we intended to transform the bacteria with. This can be done by using restriction enzymes followed by DNA gel electrophoresis. Each standard BioBrick is flanked with restriction sites, which are the target of restriction enzymes. The aim is to cut the plasmid at these sites; one cut leading to an open, linear plasmid and two cuts leading to the production of the BioBrick fragment along with the plasmid backbone. The two enzymes to be used today will be EcoRI and PstI
Protocol
Goksel about to insert some restriction enzymes into the tubes containing plasmid DNA
Although there is a protocol in Dr. Aldridge's lab explaining how to do restriction digests, Prof. Wipat came up with an alternative one. The E.coli colonies selected for further processing included cultures 2, 3, 4, 5, 6 and 7. Because 6 were chosen to be further processed, 12 Eppendorf tubes were required. In the first set of six, there would be the plasmid DNA combined with EcoRI enzyme and in the second set of six tubes, there would be plasmid DNA combined with both EcoRI and PstI.
The contents to be added to the tubes containing only EcoRI include:
- 7 microlitres of plasmid DNA
- 1 microlitre Buffer H (x10)
- 1 microlitre EcoRI
- 1 microlitre Sterile Distilled Water (SDW)
The contents to be added to the tubes containing both EcoRI and PstI include:
- 7 microlitres of plasmid DNA
- 1 microlitre Buffer H (x10)
- 1 microlitre EcoRI
- 1 microlitre PstI
Of the 12 tubes, 6 of them contained plasmid DNA along with EcorI, Buffer H and SDW whereas the other 6 contained the plasmid DNA as well as EcorI, PstI and Buffer H. This was achieved using a P20 pipette.
After this, the tubes were then lightly centrifuged (left to spin until 13,000 rpm was reached and then slowed down) and stored in the freezer.
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