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 afternoonPick colonies to start small liquid cultures growing for labWEDNESDAY 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 colonies2. Using sterile technique we will pick individual colonies from plates

Pick 2 white colonies and 1 blue colonyShake cells off the loop into 3 mL of nutrient broth + ampicillinGrow 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 now1. HARVEST BACTERIAL CELLSMove 1.5 mL of culture to tube, centrifuge at 14,000 rpm for 3 minutes to harvest bacterial cellsPull off supernatant with pipettor and put this waste into a beaker with bleach in it to kill bacterial cellsNext 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 BACTERIALyse (break open) bacterial cells to isolate plasmid DNA in cytoplasmDestroy bacterial cell wall and plasma membrane

Add 200 µL of quick lysis solution to your pelletMix tube until pellet is dissolved (resuspended) - can use vortex if neededSolution contains lysozyme which degrades cell wall and initiates cell lysisAfter pellet is resuspended incubate at room temperature for 5 min

Cloning a DNA segment from bacteriophage

Isolate plasmid from bacterial cultures

2. LYSIS OF BACTERIAAdd 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 lysisNaOH denatures DNA

NEUTRALIZATIONpH of solution is high so neutralize Add 300 µL of ammonium acetate, INVERT TUBE DO NOT VORTEX - MAY HAVE TO SHAKE TUBE GENTLYIncubate on ice for 10 min

During this step the plasmid DNA will renature but the chromosomal bacterial DNA will notThe 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 DNACentrifuge for 10 min at 14,000 rpm

Pellet = chromosomal bacterial DNA + membrane junk + proteinsSupernatant = plasmid DNA + E.coli RNAPipet supernatant into new tube

Cloning a DNA segment from bacteriophage

Isolate plasmid from bacterial cultures

4. CONCENTRATE PLASMID DNAPlasmid DNA precipitated by alcohol (ethanol, isopropanol)

To supernatant add 0.6 volumes of isopropanol (~600-700 µL)Invert tubeIncubate at room temp for 10 min - isopropanol will precipitate DNACentrifuge for 15 min at 14,000 rpm

Pull off supernatantAdd 600 µL of isopropanol and centrifuge at 14,000 rpm for 5 minPull 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 protocolPut at 37 ˚C

SEPARATE DIGESTS ON AGAROSE GEL NEXT WEEK

Cloning a DNA segment from bacteriophage

Recombinant DNA transformed into bacterial cellsSafety

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!!