1. CULTURE INDEPENDENT ANALYSIS OF MICROBIAL COMMUNITIES IN SOIL
Molecular Microbial Ecology

2. Culture-Independent Soil Analysis
Isolate DNA from soil
PCR amplify 16S rRNA genes
Agarose gel electrophoresis
Clone into vector
Transform into E. coli and selection on media
Plasmid preps
Restriction enzyme digest
Dideoxy sequencing
Sequence analysis

3. DNA Purification

4. Polymerase Chain Reaction
Used to amplify DNA in a sample
Specific primers are used to amplify a specific region of DNA – not all of it
Primers are synthetically produced to be complementary to the region of interest
Taq polymerase – DNA polymerase
Enzyme that synthesizes new DNA strands
Obtained from Thermus aqauticus
Thermophile isolated from Yellowstone
Works at 72°C

5. Melting and Annealing DNA
Melting or denaturation
Renaturation or annealing

6. PCR Repeated cycles of DNA synthesis Denaturation Annealing (Priming)
Heat to 94°C
Separate DNA strands from each other
Annealing (Priming)
Cool to 50° to 65°C
Temperature depends on Tm of primers
Primers hybridize to DNA template
Extension (Synthesis)
Heat to 72°C
DNA synthesis
Repeat the above 3 steps 20 to 40 X

7. PCR

8. Designing Thermal Cycler Protocols

9. Troubleshooting PCR Annealing temperature Touchdown annealing cycles
If not enough product, lower temp
If too much background smearing, increase temp
Touchdown annealing cycles
Increase MgCl2 concentration
Increased salts increase Tm of primer
Adjust primer concentrations

10. Agarose Gel Electrophoresis
Used to separate DNA fragments from each other
Gel is a matrix of agarose
Crosslinks with pores through which molecules can move
Electrical field is passed through the agarose gel
DNA is negatively charged and will move towards the positive pole of the gel
Smaller molecules will move faster

11. Gel Electrophoresis
To visualize the bands it is stained with a fluorescent dye which fluoresces under UV light or with methylene blue
Figure 10.4

12. Loading Gel

13. Running Gel

15. Topo TA Cloning

16. Topo TA Cloning Selection can be on LB + kanamycin or ampicillin
Ampicillin can lead to satellite colonies
If no insert, vector should not be able to close up
If it does circularize, enzyme is produced that kills cells

17. Restriction Endonucleases
Cleave double stranded DNA at specific sequences
Named for the bacteria from which they are isolated

19. DNA Sequencing
Technique used to determine exact order of nucleotides (bases) of DNA
Uses dideoxynucleotides (ddNTPs)
Lacking the 3’ hydoxyl group on the sugar
When a ddNTP is incorporated into a growing DNA strand, elongation terminates

20. DNA Sequencing

23. MNF001 and MNF003 Culture Independent Analysis