Transcript 4-2 Sources of DNA

4-2 Sources of DNA
Background
• Nature = DNA in cells
• Lab= Manipulate and grow cultures of
different cells to access DNA
– Grow in medium
– Collect and break open cells (lysis)
– Separate and isolate DNA
Genetic code is the same from organism to
organism BUT the packaging and location
Of DNA varies among organisms.
Bacterial DNA
• No nucleus or organelles (except ribosomes)
• 1 long, circular DNA (supercoiled like twisted
rubber band)
• ~1000 genes
• “Transformation” can transfer genetic
information between bacterial cells (exchange
plasmids)
• Plasmid = extra ring of DNA in cytoplasm
found in some bacteria
– 5-10 genes
– Codes for proteins that might be needed under
extreme conditions
• R-plasmid = antibiotic resistant genes
Bacterial Cell Cultures
Bacterial Cell Cultures
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Solid medium (agar plates)
Agar (algae powder) is mixed with water
Heat until suspended
Autoclave (high temp/high pressure)
Agar solution cooled to 65˚C
Pour into sterile petri dishes using sterile technique
Agar solidifies 15-20 minutes
Streak the bacteria on the plate using the “triple Z
streaking method”
Autoclave= instrument that creates
high pressure and high temperatures
to sterilize equipment and media
What are you looking at in the lab?
• If you want the DNA then you must EXTRACT
it from the bacterial cell by breaking open the
cell (lyse)
• If your product is a protein, you must also
EXTRACT the proteins by lysing the cell
• Bacterial cells are very easy to grow but must
lyse cells to get products.
Bacterial Cell Cultures
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Liquid medium (broth)
LB powder base is mixed with water
Autoclave (high temp/high pressure)
Introduce bacteria to the broth
(inoculate) using sterile technique
• Shake cultures so bacteria can grow
Eukaryotic DNA
• DNA from protists, plants, fungi and
animal cells
• Packaged into Chromosomes
– Single long linear DNA coiled around
histone proteins
– Each single DNA may contain several
million or more nucleotides and up to
many thousands of genes
– Same genetic code (4 nitrogenous bases)
as bacteria just more genes
Mammalian Cell Cultures
• More challenging to grow than
bacteria
• Mammalian cells depend on other
cells for products and stimuli for
growth
• Cells are grown in a liquid broth in
special tubes and bottles with a
bottom surface to which the cells
can stick
T-Flask
Roller Bottles
Mammalian Cell Cultures
• Media contains special nutrients
(sugars, salts, proteins) ideal for
each type of cell being grown
– Special indicators can be added to
monitor growth
• Phenol Red – pH indicator. Turns
yellow/gold as solution becomes
acidic (too much carbon dioxide
and other waste products) = cells
overgrowing!
What are you looking at in the lab?
• If you want the DNA, then you must EXTRACT
it from the mammalian cell by breaking open
the cell (lyse)
• If your product is a protein, you can EXTRACT
it from the growth media
– Mammalian cells release their proteins from the
cell into the growth media so its easy to harvest.
– Cells are unharmed!
– Mammalian cells hard to grow but easy to harvest
protein products!
Extra Stuff you might like to know
(Dr. Karkare supplements)
Characteristics of Mammalian Cells
• Require stringent environment, very shear sensitive
• Many are attachment dependent
• In addition to typical metabolic requirements, cells need growth factors
and other proteins
• Mammalian cells grow at a much slower rate than bacteria and yeast,
doubling every 14 to 70 hours
• Cell growth can be described by lag, exponential growth, then stationary
and death phases
Cell Culture Musts
• Aseptic technique
– Laminar flow hoods
– Minimize air flow disturbance
– Sterile plastic/glassware
– Use of sterile gloves etc. (humans are primary
contamination source!)
– Minimize exposure of culture to open air
Cell Culture Musts
• Appropriate culture environment
– Culture media containing glucose, amino acids,
serum (FBS or appropriate serum substitutes),
lipids etc.
– Incubation at 37o C
– pH control with bicarbonate buffer (CO2
incubator)
T-Flasks
•T-Flasks: Flat sided plastic flasks used for small
scale culturing (25 cm2 to 175 cm2
•Surface treated to generate highly charged
oxygen ions on the plastic creating negative
charges which attract most cell lines
•Other methods use chemical coatings such as
poly D-lysine
Automated Cell Culture machine at
ATCC