DNA extraction from Soil

For All you guys whom dealing with environmental molecular
For All you guys whom dealing with metagenomic world......
Relax....we have to be patient...and keep moving forward


DNA extraction from soil (Zhou et al, 1996)
Advantage: Simple & High DNA Yield......

1. Weigh 5-15 g of soil into 50-mL tube.
2. Add 13.5 mL lysis buffer (100 mM Tris-HCl, 100 mM sodium
EDTA, 100 mM sodium phosphate, 1.5 M NaCl, and 1% CTAB)
plus 100 μL of proteinase K (10 mg/mL). Shake horizontally at
37°C for 30 minutes.
3. Add 1.5 mL of 20% SDS. Incubate at 65°C for 2 hours with
gentle, end-to-end inversion every 15-20 minutes.
4. Spin at 7000 rpm (6000g) for 10 minutes.
5. Transfer supernatant to a clean 50-mL tube.
6. Add 4.5 mL lysis buffer plus 0.5 mL of 20% SDS to soil pellet.
Vortex for 10 seconds.
7. Incubate at 65°C for 10 minutes.
8. Spin at 7000 rpm (6000g) for 10 minutes.
9. Collect supernatant and combine with the supernatant from step5.
10. Repeat steps 6-9 again.
11. Extract the supernatant with equal volume of chloroform/isoamyl
alcohol (24:1 v/v).
12. Spin at 6500 rpm (5500g) for 10 minutes.
13. Collect aqueous (top) phase in a 40-mL Sorvall tube.
14. Precipitate genomic DNA with 0.6 volume of isopropanol at room
temperature for 1 hour or overnight.
15. Spin at 11,600 rpm (16,000g) for 20 minutes.
16. Discard supernatant. Wash DNA pellet with approximately 3 mL
of cold 70% ethanol. Spin at 11,600 rpm (16,000g) for 10
minutes.
17. Air-dry pellet.
18. Resuspend DNA pellet with approximately 150 μL of dH2O.

Ok good luck.......


For Detail please refer to this Article :
Zhou et. al. (1996) DNA Recovery from Soils of Diverse Composition. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. 62(2) : 316–322

DNA Extraction from Kiwifruit- Teacher Handout

Introduction

DNA is present in the cells of all living organisms. This procedure is designed to extract DNA from kiwi in sufficient quantity to be seen and spooled. This activity is ideal for students to work in pairs, but each student will have a tube of DNA at the end.

Materials

• knife for cutting kiwi
• one small ziplock bag per group of students
• jar or beaker that fits strainer or funnel
• strainer or funnel
• cheese cloth (or a #6 coffee filter)
• ice water bath (a large mixing bowl works well)
• water
• clear-colored shampoo, such as Suave Daily Clarifying Shampoo
• kiwifruit, half a kiwi per group of students
• table salt, either iodized or non-iodized
• 1 large test tube (holds 20 ml) per group, preferably with a cap
• 1 small test tube (holds 10 ml) for each student, preferably with a cap
• cold 95% ethanol (grain alcohol)

Protocol

1. Set up an ice water bath.

2. Each group will use half a kiwi and 20 ml of the following shampoo solution:
For one liter of the shampoo solution, mix 100 ml of shampoo and 15 g of table salt. Add water to make a final volume of 1 liter. Dissolve the salt y stirring slowly to avoid foaming. Measure 20 ml of solution for each group of students.

3. Peel the kiwi, cut them into about 12 pieces.

Directions for each group:

4. Get 6 pieces of kiwi and put them in a ziplock bag.

5. Add 20 ml of shampoo solution to the ziplock bag. Make sure the bag is closed with out much extra air. What do you think the shampoo solution does to the kiwi?

6. Mush the kiwi thouroughly but carefully so the bag doesn't break, for about 5 minutes. What does mushing the kiwi do?

7. Cool the kiwi mixture in the ice bath for a minute. Then mush the kiwi more. Cool, then mush. Repeat this several times. Why do we cool the mixture?

8. Filter the mixture through cheesecloth. All the groups can combine their mixtures at this point, to filter together. What is being filtered out? What is going through the filter?

9. Dispense approximately 3 ml of kiwi solution into each test tube, one for each student.

10. Being careful not to shake the tubes, add approximately 2 ml of cold 95% ethanol to each tube. What do you think the ethanol does? Why do we want it cold?

11. Take a look at your tube. What do you see in the top portion of the liquid?

Adapted from UNiv. Arizona