Cloning a DNA segment from sheep Recombinant DNA transformed into bacterial cells Last week we plated cells onto agar plates + ampicillin + X-gal Controls: E.coli-pUC18“negative control” Should only get blue colonies E.coli-pUC18-satellite“positive control” Should only get white colonies E.coli Should see NO GROWTH Your plates: Some white and LOTS of blue colonies Dr. Soukup may have tried re-streaking your one lone white colony! TUESDAY/WEDNESDAY Pick colonies to start small liquid cultures growing for your lab Wed/Thur - Isolate recombinant DNA plasmid from bacterial cells Start restriction digests, gel will be run on Dec. 3/4

Cloning a DNA segment from sheep Pick colonies to start small liquid cultures growing for tomorrow 1. Count (estimate) number of white and blue colonies 2. Using sterile technique we will pick individual colonies from plates Pick 2 white colonies and 1 blue colony Shake cells off the loop into 3-5 mL of nutrient broth + ampicillin Grow overnight at 37 ˚C DO NOT PICK SMALL “SATELLITE” COLONIES AROUND YOUR LARGE TRANSFORMANTS!! Small colonies arise because  -lactamase secreted by ampicillin-resistance gene in the large colonies that have plasmid will deplete the ampicillin in the region around the colonies Satellite colony

Cloning a DNA segment from sheep Isolate plasmid from bacterial cultures Cells in culture now 1. HARVEST BACTERIAL CELLS Move 1.5 mL of culture to a microfuge tube, centrifuge at 14,000 rpm for 3 minutes to harvest/pellet bacterial cells Pull off supernatant with pipettor and put this waste into a beaker with bleach in it to kill bacterial cells Next add another 1.5 mL of culture to the same tube and centrifuge again at 14,000 rpm for 3 minutes, repeat disposal of supernatant 2. LYSIS OF BACTERIA Lyse (break open) bacterial cells to isolate plasmid DNA in cytoplasm Destroy bacterial cell wall and plasma membrane Add 200 µL of quick lysis solution to your pellet Mix tube until pellet is dissolved (resuspended) - can use vortex if needed Solution contains lysozyme which degrades cell wall and initiates cell lysis After pellet is resuspended incubate at room temperature for 5 min

Cloning a DNA segment from sheep Isolate plasmid from bacterial cultures 2. LYSIS OF BACTERIA Add 400 µL of SDS-NaOH, INVERT TUBE DO NOT VORTEX Incubate on ice for 10 min SDS dissolves bacterial membranes and causes final stages of lysis NaOH denatures DNA NEUTRALIZATION pH of solution is currently high so need to neutralize it back down to ~7.5 Add 300 µL of ammonium acetate, INVERT TUBE DO NOT VORTEX - MAY HAVE TO SHAKE TUBE GENTLY Incubate on ice for 10 min During this step the plasmid DNA will renature but the chromosomal bacterial DNA will not The ammonium acetate and SDS cause a tangled network of chromosomal bacterial DNA and cell debris and you can separate this from smaller aqueous plasmid DNA using centrifugation 3. PURIFICATION OF PLASMID DNA Centrifuge for 10 min at 14,000 rpm Pellet = chromosomal bacterial DNA + membrane junk + proteins Supernatant = plasmid DNA + E.coli RNA Pipet supernatant into a new tube

Cloning a DNA segment from sheep Isolate plasmid from bacterial cultures 4. CONCENTRATE PLASMID DNA Plasmid DNA precipitated by alcohol (ethanol, isopropanol) To supernatant add 0.6 volumes of isopropanol ( µL) Invert tube Incubate at room temp for 10 min - isopropanol will precipitate DNA Centrifuge for 15 min at 14,000 rpm Pull off supernatant Add 600 µL of isopropanol and centrifuge at 14,000 rpm for 5 min Pull off supernatant and let air dry Resuspend pellet in 30 µL of H 2 O

Cloning a DNA segment from sheep Restriction digestion EcoR1 digestion of recombinant DNA plasmids (“B”, “W1”, “W2”) - set up as described in protocol Put at 37 ˚C Why digesting with EcoRI again? NEXT LAB: Load restriction digests on agarose gel containing ethidium bromide Examine results

Cloning a DNA segment from sheep Recombinant plasmid DNA purified from bacterial cells Agarose gel with ethidium bromide Agarose gel separates larger DNA molecules by size Ethidium Bromide fluoresces under UV light EB intercalates into DNA Put gel on UV light source after electrophoresis