1. Molecular Cloning

2. Definitions Cloning : Sub-cloning: Plasmid recombinant:
Obtaining a piece of DNA from its original source (Genome) and introducing it in a DNA vector
Sub-cloning:
Transfer a cloned DNA insert or a part thereof from one vector to another vector
Plasmid recombinant:
Vector into which foreign DNA was introduced
Recombinant organism
Organism with a recombinant vector

3. Why clone?
Separate, identify, manipulate or express a specific DNA fragment 3

4. Step 1- Separate Two approaches: Fragment/digest genomic DNA
Generates a vast number of fragments
May be difficult to find fragment of interest
PCR Amplification
Much less fragments
Much easier to find sequence of interest

5. Cloning Generate compatible ends DNA ligation Restriction Enzyme
Appropriate vector
Fragment of interest
DNA Recombination
Intermolecular ligation
Recombinant
Intramolecular ligation
Non-Recombinant
Transformation
Host Cells
Recombinant cell
Non-Recombinant cell
1 plasmid/1 cell

6. Amplification of Recombinant Plasmids
Duplication
1 colony= 1 clone with 1 plasmid + 1 insert
Bacterial growth

7. Screening or Identification of Recombinant Plasmid clones
Restriction mapping
Hybridization
PCR

8. Expression Why? Produce the protein in a heterologous system
Produce large quantities of the protein
Purify the protein
Study the protein’s activity

9. Amplification, Cloning & Expression of LacZ– Project II
Part I
PCR amplification & mutagenesis of LacZ
Part II
Cloning of LacZ in pUC19
Part III
Enzyme assays of recombinant proteins

10. Purification & Digestion of LacZ Amplicon
Purification by QiaQuick
Removes buffer, salts, etc.
Removes primers
Removes enzyme
Digestion
Restriction sites NdeI and PstI were added to amplicon sequence
Not present in LacZ sequence
Unique in pUC19 vector
Allows directional cloning