cloning a dna segment from sheep recombinant dna transformed into bacterial cells last week we...

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.coliShould see NO GROWTH

Your plates:Some white and LOTS of blue coloniesDr. Soukup may have tried re-streaking your one lone white colony!

TUESDAY/WEDNESDAYPick colonies to start small liquid cultures growing for your labWed/Thur - Isolate recombinant DNA plasmid from bacterial cells

Start restriction digests, gel will be run on Dec. 3/4


Pick colonies to start small liquid cultures growing for tomorrow

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 3-5 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 the large colonies that have plasmid will deplete the ampicillin in the region around the colonies

Satellite colony


Isolate plasmid from bacterial cultures

Cells in culture now1. HARVEST BACTERIAL CELLSMove 1.5 mL of culture to a microfuge tube, centrifuge at 14,000 rpm for 3 minutes to harvest/pellet bacterial cellsPull off supernatant with pipettor and put this waste into a beaker with bleach in it to kill bacterial cellsNext 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 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



2. LYSIS OF BACTERIAAdd 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 lysisNaOH denatures DNA

NEUTRALIZATIONpH of solution is currently high so need to neutralize it back down to ~7.5Add 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 a new tube



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


Restriction digestion

EcoR1 digestion of recombinant DNA plasmids (“B”, “W1”, “W2”) - set up as described in protocolPut at 37 ˚C

Why digesting with EcoRI again?

NEXT LAB:Load restriction digests on agarose gel containing ethidium bromideExamine results


Recombinant plasmid DNA purified from bacterial cells

Agarose gel with ethidium bromideAgarose gel separates larger DNA molecules by sizeEthidium Bromide fluoresces under UV lightEB intercalates into DNAPut gel on UV light source after electrophoresis

cloning a dna segment from sheep recombinant dna transformed into bacterial cells last week we...

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