In vivo cloning

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Stage 1:	Attach the target gene to a vector e.g. a plasmid, using DNA ligase and restriction endonuclease.
1.	Isolate the vector from a bacteria.
2.	The vector is cut using the same restriction endonuclease as separating the target gene, so that the sticky ends are complementary.
3. Ligation	Mix the target gene and the vector together with DNA ligase which joins the two sticky ends together.
4.	This new vector and target gene combination is known as a recombinant DNA.
Stage 2:	Transforming the vector to the bacteria.
1.	You give a heat shock to the bacteria to create pores in its membrane. Then you add the vectors.
2.	The vector does its job by inserting itself into the bacteria.
3.	The new bacteria is known as the transformed cell.
4.	Only about 5% of cells get transformed so this is quite a unreliable process.
Stage 3:	Identifying the transformed cells.
1.	Add a marker gene to the vector before insertion.
2.	Add the bacteria solution to a agar plate.
3.	Shine UV light.
4.	Any transformed cells will fluoresce.
Stage 4:	Making a protein
1.	Attach a promoter and a terminator to the target gene.
2.	The promoter allows RNA polymerase to bind.
3.	So the target gene can be transcribed.
4.	The transformed cells are also allowed to divide to produce lots of them by incubation.

Zusammenfassung der Ressource

	Erstellt von Jumael Zafar vor fast 7 Jahre