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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
Should only get blue colonies
E.coli-pUC18-bacteriophage
Should only get white colonies
“negative control”
“positive control”
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!!