Cloning Genes

Many recombinant DNA methods require numerous copies of a specific DNA fragment. One way to obtain these copies is to place the fragment in a bacterial cell and allow the cell to replicate the DNA. This procedure is termed gene cloning, because identical copies (clones) of the original piece of DNA are produced.

DNA is cleaved by restriction enzymes and transferred to an agarose gel. The fragments are separated by gel electrophoresis.

^ The gel is soaked in an alkali solution to denature the double-stranded DNA and then placed on a platform in a dish containing buffer.

DNA is cleaved by restriction enzymes and transferred to an agarose gel. The fragments are separated by gel electrophoresis.

^ The gel is soaked in an alkali solution to denature the double-stranded DNA and then placed on a platform in a dish containing buffer.

Weight

Weight

A membrane is positioned on top of the gel.

Nitrocellulose or other membrane

Blotting paper paper passes through the gel, carrying DNA onto the membrane.

Platform

Blotting paper

A membrane is positioned on top of the gel.

Nitrocellulose or other membrane

Blotting paper

Platform

Buffer drawn up into ' the top layer of blotting paper passes through the gel, carrying DNA onto the membrane.

Membrane DNA

Membrane DNA

DNA on the membrane is fixed,.

^ .placed in a hybridization bottle with solution that contains a radioactively labeled probe, and gently rotated.

DNA on the membrane is fixed,.

^ .placed in a hybridization bottle with solution that contains a radioactively labeled probe, and gently rotated.

-Radioactive probe

Vn The probe binds to complementary DNA fragments on the membrane,.

Autoradiography -*

^ .and autoradiography detects fragments with probe attached.

Size standards

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