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 25 o C. Incubate samples from your body or an animal at 37 o C. 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 l solution S1; vortex 15 seconds. S1 contains SDS; ionic detergent disrupts lipids in membrane & promotes lysis. 4. Add 200 l 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 l supernatant to clean 2-ml tube. 8. Add 250 l solution S2; vortex 5 seconds. Incubate 4 o C 5 minutes. S2 precipitates proteins. 9. Centrifuge tube 1 minute at 10,000 x g. 10. Transfer 700 l 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 = (R) (rpm/1000) 2 R = radius in cm = ~ 8 cm for Eppendorf centrifuge 10,579 rpm = 10,000 x g 10,000 rpm = 8,936 x g 13,200 rpm = 15,570 x g

11. Load 700 l 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 l 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 l 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 l 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 -20 o C until class 13.

Use yellow tip to remove ethanol trapped on rim above filter before you elute DNA.