1. 4-2 Sources of DNA

2. 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.

3. 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

4. Bacterial Cell Cultures

5. Bacterial Cell Cultures
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 minutes
Streak the bacteria on the plate using the “triple Z streaking method”

6. Autoclave= instrument that creates high pressure and high temperatures to sterilize equipment and media

7. 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.

8. Bacterial Cell Cultures
2) 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

9. 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

10. 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

11. T-Flask
Roller Bottles

12. 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!

13. 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!

14. Extra Stuff you might like to know (Dr. Karkare supplements)

15. Characteristics of Mammalian Cells
Moorpark College STEP Workshop April 17, 2010
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
Take off “mammals”
Stem Cells and Biotechnology

16. 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

17. 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)

18. 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

19. Automated Cell Culture machine at ATCC