1. DEPARTMENT OF MICROBIOLOGY, S.M. JOSHI COLLEGE, HADAPSAR, PUNE
Cloning Genes BY
NIKAM C.D.
ASSISTANT PROFESSOR,
DEPARTMENT OF MICROBIOLOGY,
S.M. JOSHI COLLEGE, HADAPSAR, PUNE

2. Cloning Genes
Gene cloning: amplifying a specific piece of DNA via a bacteria cell
Cloning vector: a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell
Has features that make it easier to insert DNA and select for presence of vector in cell.
Origin of replication
Antibiotic resistance gene
Cloning site

3. 19.5 An idealized cloning vector has an origin of replication, one or more selectable markers, and one or more unique restriction sites.

4. Cloning Genes Plasmid vectors
Linkers: synthetic DNA fragments containing restriction sites
Transformation of host cells with plasmids
Selectable markers are used to confirm whether the cells have been transformed or not.

5. 19. 6 The pUC19 plasmid is a typical cloning vector
19.6 The pUC19 plasmid is a typical cloning vector. It contains a cluster of unique restriction sites, an origin of replication, and two selectable markers— an ampicillin-resistance gene and a lacZ gene.

6. 19.7 A foreign DNA fragment can be inserted into a plasmid with the use of restriction enzymes.

7. 19.8 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin- resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

8. 19.8 (part 2) The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin-resistance gene. [Photograph: Cytographics/Visuals Unlimited.]

10. TA cloning of PCR product

11. Bacterial transformation
Use modified strain of E. coli
Restriction system knocked out
Recombination system limited
LPS is truncated
Pores are made in cell membrane by treatment with calcium chloride.
Cells must be kept cold to prevent pores from closing

12. Bacterial transformation
DNA added to cells forms intimate interaction with membrane
Briefly moved to 42° C to heat shock cells
Stresses them and causes them to take up DNA

13. Thank you