Laboratory: Unit 3: isolate bacteria (pages 43-52)
Unit 4: extract DNA (pages 71-83)
Next Day: examine plates & streak
Lecture: DNA extraction from bacterial populations
In-Class Writing: answer questions (page 41)
Hand In: flow chart 4, first day (DNA extraction)
Read: unit 3 (manual, pages 43-83); unit 4 (71-83)
Due Next Class: lab report 2 draft (pages 37-39)
Sample Collection for Units 3 & 5 (Class 7)
1. Label agar plate & Bead Solution tube with seat
number.
2. Collect sample with sterile swab.
For dry surfaces, wet swab with sterile water.
For soil, add 0.25 g directly to Bead Solution tube.
Remove swab from container, collect sample, streak
once on agar.
Next, insert swab into Bead Solution tube; swirl
vigorously 15 seconds.
Return to lab; complete streaking with sterile
toothpicks.
3. Incubate plate at appropriate temperature.
Bacteria from soil or water probably grow best at
room temperature or 25oC.
Incubate samples from your body or an animal at
37oC.
Incubate plate overnight and examine next day.
4. One day after class 6:
Describe bacterial & fungal colonies.
Note color, shape, size, abundance of each type.
Choose two different isolated bacterial colonies.
Streak to produce single colonies.
Incubate overnight at proper temperature.
Two days after class 6: TAs move streaked plates to
cold room.
Handle cultures as though they are human
pathogens.
Avoid contact and autoclave cultures, supernatant
solutions, contaminated tubes, pipettes, tips.
Class 8: inoculate broth (p. 52, Sec. A, step 5) &
microscopy (pages 52-53).
Extract DNA from Uncultured Bacterial Community
Unit 4 (Class 7)
Wear clean gloves; use aerosol-resistant tips.
1. Collect sample with sterile BD BBL Culture
Swab EZ.
For dry surface, wet swab with sterile water.
For soil, add 0.25 g directly to Bead Solution tube.
Insert swab into Bead Solution tube;
swirl vigorously 15 seconds.
Remove swab from Bead Solution tube; secure lid.
2. Vortex 15 seconds.
3. Add 60 ml solution S1; vortex 15 seconds.
S1 contains SDS; ionic detergent disrupts lipids in
membrane & promotes lysis.
4. Add 200 ml of solution IRS
Inhibitor Removal Solution precipitates humic acid
& other PCR inhibitors.
5. Secure bead tubes horizontally to vortex platform.
Vortex at maximum speed 10 minutes.
Bead beating causes mechanical lysis.
6. Centrifuge tubes 10,000 x g (10,500 rpm) 30 sec.
7. Transfer 500 ml supernatant to clean 2-ml tube.
8. Add 250 ml solution S2; vortex 5 seconds.
Incubate 4oC 5 minutes.
S2 precipitates proteins.
9. Centrifuge tube 1 minute at 10,000 x g.
10. Transfer 700 ml supernatant to clean 2-ml
centrifuge tube containing 1.3 ml solution S3.
Vortex 5 seconds.
S3 contains high salt; allows DNA to bind silica.
RCF = 11.17 (R) (rpm/1000)2
R = radius in cm = ~ 8 cm
for Eppendorf centrifuge
RCF @ 10,579 rpm = 10,000 x g
RCF @ 10,000 rpm = 8,936 x g
RCF @ 13,200 rpm = 15,570 x g
11. Load 700 ml DNA/S3 mix into spin filter
cartridge; centrifuge 10,000 x g 1 minute.
Discard flow through; repeat until all DNA
solution is filtered (3 loads/sample).
12. Add 300 ml solution S4; centrifuge 30 seconds
10,000 x g. Discard flow through.
S4 contains ethanol; washes impurities from DNA
bound to silica filter.
Repeat wash with 300 ml S4.
Discard flow through.
13. Centrifuge 1 min. 10,000 x g to remove ethanol
completely. Use yellow tip to remove drops of
ethanol from rim (inside cartridge) above filter.
14. Place cartridge in clean tube; add 50 ml solution
S5 (10 mM Tris, pH 8.0) to center of filter. Let
stand 1 minute. Centrifuge 30 sec.10,000 x g. S5
lacks salt; releases DNA from silica filter.
15. Discard filter cartridge. Secure tube lid; write
seat number on tube.
Freeze DNA at -20oC until class 13.
Use yellow tip
to remove ethanol
trapped on rim
above filter before
you elute DNA.