Cloning a DNA segment from bacteriophage
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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

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Cloning a dna segment from bacteriophage

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

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

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

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

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

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

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!!


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