Bio-rad Ligation and Transformation Module Manuel d'utilisateur télécharger pdf (Page 24)

Possible ligation products.

Transformation

Once a gene or part of a gene has been amplified using PCR and ligated

into a plasmid, the next step in cloning is transformation, introducing the

plasmid into living bacterial cells so that it can be replicated. Heat shock

transformation and electroporation are the two methods of bacterial

transformation commonly used in the laboratory. Both methods require

competent cells, bacterial cells that can take up DNA. Not all cells are

naturally competent. For example some species, such as

Bacillus subtilis

can be easily transformed, but for other species, such as

Escherichia coli

only a small number of cells in a culture may be able to take up DNA.

Competent cells may be prepared in the laboratory or purchased

commercially.

PCR Fragment

No interruption of

lethal gene so

transformed

bacteria will die

Can be minimized

by controlling

molar ratio of

inserts

Product cannot

replicate

Desired product –

insert can be in

either orientation

Self-ligation

of vector

Multiple

inserts

Self-ligation

of inserts

Ligation

of vector

and insert

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Bio-rad Ligation and Transformation Module Manuel d'utilisateur Page 24