Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Preparation of X-gal plates - by Dr. Soukup before lab Preparation of competent cells - by Dr. Soukup before lab DURING LAB: Electrophoretic analysis of restriction digests Transformation of recombinant plasmid into bacteria Plating of bacteria onto agar plates + ampicillin + X-gal

Cloning a DNA segment from bacteriophage lambda Electrophoretic analysis of restriction digests Receive agarose gel with ethidium bromide - done by Dr. Soukup Load restriction digests on gel with size standards Examine results Agarose gel separates larger DNA molecules by size Ethidium Bromide fluoresces under UV light EB intercalates into DNA Put gel on UV light source after electrophoresis

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Transformation of recombinant plasmid into bacteria (cells that take up plasmid are “transformed”)

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Plasmid characteristics - small circular double-stranded DNA, usually not necessary for survival BUT can carry genes that confer resistance to antibiotics or allow survival in certain environments

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Plasmid characteristics Ampicillin: antibiotic used to kill bacteria by interfering with synthesis of bacterial cell wall and leads to lysis of bacteria Ampicillin is a broad-spectrum semi-synthetic penicillin that will kill gram-negative and gram- positive bacteria, includes E.coli and Salmonella E.coli in nature can become resistant to ampicillin by taking up plasmids that contain Amp-resistant genes Today we will make one of these plasmids - ampicillin resistance gene codes for Beta-lactamase (penicillinase) that inactivates (degrades) ampicillin

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Making of competent cells Treat bacterial cells (E. coli strain DH5  ) with CaCl 2, which will make them COMPETENT to take up plasmid DNA (plasmid DNA will enter the cell) CaCl 2 causes small holes to form in the cell membrane that DNA can then traverse through We have pre-made competent cells LABORATORY PROCESS TO MAKE COMPETENT CELLS: Grow small culture of bacterial from a single colony overnight at 37 ˚C Next day use small culture to seed large culture and grow to mid-log phase growth Wash the cells with CaCl 2 Incubate the cells at 4 ˚C for 12 hours

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Making of competent cells Bacterial growth in liquid media Exponential growth occurs until no O 2 left Measure cell growth by: cell count (microscope) cell mass (A 600 ) < 90 min Doubling time = 20 min

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Transformation procedure 1. Cells + plasmid DNA - incubate on ice for 20 min (cells starting to take up plasmid) DO NOT VORTEX OR ROUGHLY FLICK TUBE WITH CELLS - THEY ARE VERY FRAGILE 2. Transfer tube to 37 ˚C for 5 min (heat shock - causes faster uptake of plasmid) 3. Add nutrient broth (media) without ampicillin and incubate at 37 ˚C for 45 min USE STERILE TECHNIQUES!!!! MUST HAVE FLAME ON!!! During this 45 min the plasmid has time to start expressing the amp-resistance gene and the bacteria cell recovers from CaCl 2 treatment (repairs its membrane)

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Transformation procedure 4. Plate cells onto agar plates + ampicillin + X-gal USE STERILE TECHNIQUES!!!! MUST HAVE FLAME ON!!! You will be spreading your bacteria onto the agar plate using a glass rod Pipet culture onto plate and then spread using STERILE TECHNIQUES!! Dr. Soukup will demonstrate how to produce single colonies of control plasmids

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Transformation procedure 4. Plate 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

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Transformation procedure 4. Plate cells onto agar plates + ampicillin + X-gal Plasmid also has Lac Z gene which codes for  -galactosidase (hydrolyzes lactose, other  -galactosides and X-gal) X-gal is 5-bromo-4-chloro-3-indolyl-  D galactoside - chromogenic substrate because its product is colored X-gal converted to blue product by  -galactosidase pUC18 has a portion of Lac Z gene, remaining portion is encoded by E.coli strain SO when cells transformed with pUC18 Complementation occurs and cells make active  -gal Active  -gal causes blue colonies to be produced on agar with X-gal CAN DETERMINE WHICH PLASMIDS HAVE FOREIGN DNA Polylinker is inserted in Lac Z gene - if no foreign DNA inserted then  -gal made and colonies BLUE If foreign DNA (bacteriophage DNA) inserted - complementation is destroyed and no active  -gal, so colonies WHITE

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Transformation procedure 4. Plate cells onto agar plates + ampicillin + X-gal Possible reasons for WHITE COLONIES??? RESTRICTION DIGESTS RECIRCULARIZATION OF pUC18 during ligation with no foreign DNA inserted

Cloning a DNA segment from bacteriophage lambda Recombinant DNA transformed into bacterial cells Safety 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!!