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Cloning a DNA segment from bacteriophage. 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-bacteriophage “positive control”
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Cloning a DNA segment from bacteriophage 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-bacteriophage “positive control” Should only get white colonies Your plates: Some white and blue colonies? Dr. Soukup may have had to re-streak some of the white colonies TUESDAY afternoon Pick colonies to start small liquid cultures growing for lab WEDNESDAY lab - Isolate recombinant DNA plasmid from bacterial cells Start restriction digests for next week
Cloning a DNA segment from bacteriophage Pick colonies to start small liquid cultures growing for WED lab 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 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 those colonies that have plasmid will deplete the ampicillin in the region around the colonies Satellite colony
Cloning a DNA segment from bacteriophage Isolate plasmid from bacterial cultures Cells in culture now 1. HARVEST BACTERIAL CELLS Move 1.5 mL of culture to tube, centrifuge at 14,000 rpm for 3 minutes to harvest bacterial cells Pull off supernatant with pipettor and put this waste into a beaker with bleach in it to kill bacterial cells Next add rest 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 bacteriophage Isolate plasmid from bacterial cultures 2. LYSIS OF BACTERIA Add 400 µL of SDS-NaOH, INVERT TUBE DO NOT VORTEX - CELLS/DNA FRAGILE!! Incubate on ice for 10 min SDS dissolves bacterial membranes and causes final stages of lysis NaOH denatures DNA NEUTRALIZATION pH of solution is high so neutralize 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 new tube
Cloning a DNA segment from bacteriophage Isolate plasmid from bacterial cultures 4. CONCENTRATE PLASMID DNA Plasmid DNA precipitated by alcohol (ethanol, isopropanol) To supernatant add 0.6 volumes of isopropanol (~600-700 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 H2O
Cloning a DNA segment from bacteriophage Restriction digestion EcoR1 digestion of recombinant DNA plasmids (“B”, “W1”, “W2”) - set up as described in protocol Put at 37 ˚C SEPARATE DIGESTS ON AGAROSE GEL NEXT WEEK
Cloning a DNA segment from bacteriophage Recombinant DNA transformed into bacterial cells Safety LIQUID WASTE MUST BE TREATED WITH BLEACH!! WASH YOUR HANDS WITH SOAP!!!! DISINFECT LAB BENCH WITH BLEACH OR ETHANOL SOLUTION IF YOU SPILL BACTERIA TELL DR. SOUKUP LIMIT EXPOSE OF BACTERIA TO AIR PLACE ALL BACTERIAL WASTE IN RED BIOHAZARD BAGS WEAR GLOVES!!
