Methods of transformation Electroporation Electrical shock makes cell membranes permeable to DNA Makes holes in cells Calcium Chloride/Heat-Shock Chemically-competent cells uptake DNA after heat shock We will do this method

Bacterial Transformation Look at figure 5.7 Competent E. coli are able to take up the plasmid Transfer plasmid DNA into bacteria Transfer to selective media Grow overnight Pick colonies – miniprep or chromatography

Transformation procedure Suspend bacterial colonies in Transformation solution Add S3 or pGLO plasmid DNA Put cells in CaCl2 = Ca ++ binds to PO4- Place tubes on ice Heat-shock at 42°C and place on ice Incubate with nutrient broth Streak plates

Bacterial Transformation Cell wall GFP Bacterial chromosomal DNA Beta lactamase (ampicillin resistance) pGLO plasmids

Transformation Procedure Overview Day 1 Day 2

Selection Plated transformed cells on antibiotic – Ampicillin Only a small % of the cells will undergo transformation Those cells that grow contain the plasmid Remember, Arabinose causes cells to express GFP and grow green

Recombinant DNA = plasmid + insert How do you know if insert is present? Restriction digest Blue/White selection (figure 5.10) pJET1.2plasmid (figure 5.11)

Blue/White Selection (Fig. 5.10) If fragment is not inserted, bacterial cells will be blue If fragment is inserted and LacZ is disrupted, bacterial cells will be white LacZ = b-galactasidase cleaves artificial substrate X gal to produce blue colonies

pJET1.2 plasmid (fig 5.11) Ec0471R is toxic If ligate a fragment before Ec0471R, you will inactive the gene and bacterial cells will grow. If there is no insert, Ec0471R will be toxic and the bacterial cells will die.

Transformation efficiency Page 143 – 144 We will look back at these calculations after we perform our transformations.