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 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 4oC 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 = 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 gRCF @ 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 -20oC until class 13.

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