DNA Technology BACTERIAL TRANSFORMATION REVIEW Ms Day Honors

DNA Technology: BACTERIAL TRANSFORMATION REVIEW Ms. Day Honors Genetics

Gene Cloning 1. Find plasmid & gene of interest you want to use 2. Cut (digest) both DNA’s with SAME restriction enzyme • Why? To make SAME sticky ends!!! 3. Put both samples together in a medium (liquid) 4. Add DNA ligase • Why? To seal up plasmid’s DNA backbone 5. Now…you have a recombinant plasmid ready for transformation!

Vocab Review: Gene of Interest (Foreign DNA) Recombinant Plasmid Cloning Vector

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What is Bacterial Transformation? • Transformation – “Naked” Plasmids (present in environment) are taken up by certain bacteria – Viruses are NOT used in this method!

More Vocab Review

Bacterial Transformation • Step 1 DNA Isolation – Isolation of the “Gene of Interest” (foreign DNA) • Step 2: Gene Cloning/Make Recombinant DNA – Insertion of foreign DNA into bacterial plasmid using restriction enzymes and DNA ligase • http: //www. dnalc. org/resources/animations/transformation 1. html • Step 3 Transformation – Insertion of recombinant DNA into bacteria by making bacteria competent (weaken) • Use Ca. Cl 2 and heat shock techniques

How do you make Bacteria competent? • Step 1: Add Calcium Chloride (Ca. Cl 2) – Ca. Cl 2 is in a solution (creates Ca+2 and Cl- ions) – DNA in plasmid is negatively charged due to phosphate groups in the backbone – Cell membrane of E. coli also is negatively charged because phospholipids are made of same phosphate groups (PO 4 -3) – Ca+2 ions neutralize charges so plasmid can get near (and inside) bacterial cell.

How do you make Bacteria competent? • Step 2: Use Heat Shock – Heat Shock is a process that uses warm water (bath) and ice to help get plasmid inside cell • Add recombinant plasmid + host cell + Ca. Cl 2 solution to ice then heat then back on ice – Heat = increases kinetic energy of matter • Molecules/atoms move faster – Ice = decreases kinetic energy of matter • Molecules/atoms move slower • http: //www. dnalc. org/resources/animations/transformati on 2. html

What is a selective marker? • A gene added to a recombinant plasmid IN ADDITION to the gene of interest • Selective markers are usually genes that allow bacteria to be resistance to antibiotics • Antibiotic KILL bacteria by breaking down its cell wall. • Ex: amp. R (ampicillin resistance gene) • Used to determine if a cell is transformed or not

GFP Protein • https: //www. youtube. com/watch? v=E 7 HQB dv 0 Ebo (3: 41) • https: //www. youtube. com/watch? v=wxf 4 a 4 SX 84 A (5: 41) • https: //www. youtube. com/watch? v=x 5 ox 71 q. Ia-0