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Method |
Comments |
| 1) The DNA vector is cut with one or two restriction enzymes. | The carefully chosen enzyme cuts the plasmid in the Multiple Cloning Site only once. The Multiple Cloning Site is situated in the middle of the Lac gene, which codes for the galactosidase. |
| 2) The Insert is cut with the same enzyme (s) as the vector | The insert and the vector will be able to stick to one another. This concept is better explained in the chapter about Ligation |
| 3) The Insert is ligated to the plasmid. | In this operation, there is a certain level of probability that the plasmid will ligate onto itself. This can be avoided when using two enzymes to cut the vector, but there is still a remote chance for it to happen. |
| 4) The construct 'Vector / Insert' is introduced into a bacterium. This is called transformation. | You have to understand that we have a solution with thousands of constructs, and some might not have worked. The plasmid might not have been digested properly, or the insert might not be there. In the same way, we have more than one bacterium. It is a solution of many bacteria we mix with a solution of many constructs. |
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5) The solution construct / bacterium is spread onto a agar plate containing
Ampicillin. Some XGal and Inducer are introduced at this stage.
The plates are left at 37°C for 18 hours. |
When the solution is spread onto the plates, the bacteria will be isolated.
They will grow "on the spot", and a single colony will be a clone
of the same original one. If the original one has a plasmid with an insert,
it will be white. If the bacteria has a plasmid without an insert it will
be blue.
Bacteria without plasmids do not grow |
| 6) A single white colony is picked up, grown in a liquid medium and plasmid DNA is extracted. | This step can be performed in many different ways, but they are all very similar to the method we review in the chapter about the preparation of DNA . |
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