1. Molecular Cloning

2. Definitions   Cloning :   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   Recombinant Plasmid :   Vector into which foreign DNA was introduced   Recombinant organism   Organism with a recombinant vector

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

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. Restriction analysis of digested genomic DNA   Southern analysis   Separate DNA fragments according to size   Denature   Hybridize with single stranded probe representing region of interest

6. Southern Agarose gel Probe Hybridize to probe

7. 1 plasmid/1 cell DNA Recombination Restriction Enzyme Restriction Enzyme Transformation Host Cells Fragment of interest Appropriate vector Generate compatible ends DNA ligation Intermolecular ligation Recombinant Intramolecular ligation Non-Recombinant Cloning Recombinant cellNon-Recombinant cell

8. 1 colony= 1 clone with 1 plasmid + 1 insert Plasmid Duplication Bacterial growth Amplification of Recombinant Plasmids

9. Screening and Identification of Recombinant Plasmid clones   Restriction mapping   Hybridization   PCR

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

11. Amplification, Cloning & Expression of GFP – Project II   Part I   PCR amplification & mutagenesis of GFP   Part II   Cloning in pGFPuv   Part III   Enzyme assays of recombinant proteins

12. Purification & Digestion of GFP Amplicon   Purification by QiaQuick   Removes buffer, salts, etc.   Removes primers   Removes enzyme   Digestion   Restriction sites XhoI and HindIII are present in amplicon sequence   Allows directional cloning

13. Cloning strategy   Substitute wild type GFP fragment for mutated one XSH WT GFP XSH Mutated GFP