**Fungal Spore gDNA Preparation from Soil_RevC**

Prepared by: Todd DeSantis

Revision Date: April 18, 2006

+----+-------------------------------+---------------------------------+
| ** | **Action**                    | **Notes**                       |
| St |                               |                                 |
| ep |                               |                                 |
| ** |                               |                                 |
+----+-------------------------------+---------------------------------+
| *  |                               |                                 |
| So |                               |                                 |
| il |                               |                                 |
| Di |                               |                                 |
| sr |                               |                                 |
| up |                               |                                 |
| tt |                               |                                 |
| io |                               |                                 |
| n* |                               |                                 |
+----+-------------------------------+---------------------------------+
| 1  | Obtain 2mL tube with 3 bead   |                                 |
|    | silica mix.                   |                                 |
+----+-------------------------------+---------------------------------+
| 2  | Add 550 uL MoBio Bead         |                                 |
|    | Solution                      |                                 |
+----+-------------------------------+---------------------------------+
| 3  | Add 100 mg soil sample and    |                                 |
|    | vortex 5s.                    |                                 |
+----+-------------------------------+---------------------------------+
| 4  | Add 60 uL MoBio S1, invert to |                                 |
|    | mix.                          |                                 |
+----+-------------------------------+---------------------------------+
| 5  | Add 200 uL MoBio IRS          | Inhibitor Removal Solution      |
+----+-------------------------------+---------------------------------+
| 6  | Disrupt in FP120, 6.5m/s, for | Haven\'t tested if no ice make  |
|    | 2 min, in 40s intervals.      | a difference.                   |
+----+-------------------------------+---------------------------------+
| 7  | Spin 10k\*g 1 min.            |                                 |
+----+-------------------------------+---------------------------------+
| 8  | Remove 500 uL super. to       |                                 |
|    | Falcon 2509.                  |                                 |
+----+-------------------------------+---------------------------------+
| 9  | Add 225 uL Bead Solution, 30  | This allows further extraction  |
|    | uL S1, and 200 uL IRS.        | of the lysate pellet.           |
|    |                               | Empirically found that majority |
|    |                               | of DNA is not in the initial    |
|    |                               | lysate supernatant.             |
+----+-------------------------------+---------------------------------+
| 10 | Vortex at various angles      |                                 |
|    | until beads are resuspended.  |                                 |
|    | (appox. 10s)                  |                                 |
+----+-------------------------------+---------------------------------+
| 11 | Vortex 20s tapped             |                                 |
|    | horizontally.                 |                                 |
+----+-------------------------------+---------------------------------+
| 12 | Spin 1 min 10k\*g.            | 10,000 RPM in BiofugePico,      |
|    |                               | 11,000 RPM in Spectrafuge       |
+----+-------------------------------+---------------------------------+
| 13 | Remove 500 uL super. to 2 mL  |                                 |
|    | microtube.                    |                                 |
+----+-------------------------------+---------------------------------+
| 14 | Repeat steps 9 -13.           | After this third supernatant is |
|    |                               | obtained you should have 1.5 mL |
|    |                               | total.                          |
+----+-------------------------------+---------------------------------+
| 15 | Spin supernatant pool,        | Further clarify lysate          |
|    | 16k\*g, 10\'                  | supernatant.                    |
+----+-------------------------------+---------------------------------+
| 16 | Transfer clarified            | polypropylene to withstand the  |
|    | supernatant to new tube.      | phenol/chloro                   |
+----+-------------------------------+---------------------------------+
| *  |                               |                                 |
| Ph |                               |                                 |
| en |                               |                                 |
| ol |                               |                                 |
| E  |                               |                                 |
| xt |                               |                                 |
| ra |                               |                                 |
| ct |                               |                                 |
| io |                               |                                 |
| n* |                               |                                 |
| (C |                               |                                 |
| ur |                               |                                 |
| re |                               |                                 |
| nt |                               |                                 |
| Pr |                               |                                 |
| ot |                               |                                 |
| oc |                               |                                 |
| ol |                               |                                 |
| s, |                               |                                 |
| Au |                               |                                 |
| su |                               |                                 |
| be |                               |                                 |
| l) |                               |                                 |
+----+-------------------------------+---------------------------------+
| 17 | Add 1.5 mL                    | Equal volume to clarified       |
|    | phenol/chloroform/isoamyl     | supernatant. If it\'s pinkish,  |
|    | alcohol.                      | then it has oxidized and may    |
|    |                               | damage nick DNA. To prevent     |
|    |                               | this, place nitrogen in the     |
|    |                               | head space after each use and   |
|    |                               | store at 4C.                    |
+----+-------------------------------+---------------------------------+
| 18 | Vortex 10s                    |                                 |
+----+-------------------------------+---------------------------------+
| 19 | Spin 1200 g, 5 min room temp. |                                 |
+----+-------------------------------+---------------------------------+
| 20 | Note if a white precipitate   |                                 |
|    | was at the interface and      |                                 |
|    | transfer aq. layer to new     |                                 |
|    | tube.                         |                                 |
+----+-------------------------------+---------------------------------+
| 21 | Add 200 uL TE to organic      | Recovers more nucleic acid from |
|    | layer, vortex and spin as     | organic phase.                  |
|    | above.                        |                                 |
+----+-------------------------------+---------------------------------+
| 22 | Transfer aq. layer to same    |                                 |
|    | tube as in step 20.           |                                 |
+----+-------------------------------+---------------------------------+
| 23 | If a white precipitate was at | White precipitate is protein.   |
|    | the interface, add another    | Repeat these extractions until  |
|    | 1.5 mL P/C/I to aqueous       | no more protein is seen at the  |
|    | solution, vortex 10s, and     | interface.                      |
|    | spin as before, transfer aq.  |                                 |
|    | layer.                        |                                 |
+----+-------------------------------+---------------------------------+
| 24 | Add 2 mL chloroform, vortex   | To remove residual phenol.      |
|    | 10s                           |                                 |
+----+-------------------------------+---------------------------------+
| 25 | Spin 1200 g, 5 min R.T.       |                                 |
+----+-------------------------------+---------------------------------+
| 26 | Transfer aq. layer to new     |                                 |
|    | tube Falcon 2509              |                                 |
+----+-------------------------------+---------------------------------+
| *E |                               |                                 |
| th |                               |                                 |
| an |                               |                                 |
| ol |                               |                                 |
| Pr |                               |                                 |
| ec |                               |                                 |
| ip |                               |                                 |
| it |                               |                                 |
| at |                               |                                 |
| io |                               |                                 |
| n* |                               |                                 |
| (P |                               |                                 |
| et |                               |                                 |
| er |                               |                                 |
| A  |                               |                                 |
| gr |                               |                                 |
| on |                               |                                 |
| a  |                               |                                 |
| nd |                               |                                 |
| Au |                               |                                 |
| su |                               |                                 |
| be |                               |                                 |
| l) |                               |                                 |
+----+-------------------------------+---------------------------------+
| 27 | Add 5mg/mL linear acrylamide  | final concentration between 10  |
|    | (Ambion).                     | to 20 ug/mL (concentration      |
|    |                               | after addition of EtOH and      |
|    | Use 1.3% of the volume of     | sodium acetate) \*\*\*see       |
|    | your aq solution.             | calculations below...           |
+----+-------------------------------+---------------------------------+
| 28 | Add 1/10 volume 3M sodium     | 1/10 of (aq. volume +           |
|    | acetate pH 5.2, vortex 5s.    | acrylamide added)               |
+----+-------------------------------+---------------------------------+
| 29 | Add 3 volumes ice cold 100%   |                                 |
|    | EtOH.                         |                                 |
+----+-------------------------------+---------------------------------+
| 30 | Chill -20C for over 30 min.   |                                 |
+----+-------------------------------+---------------------------------+
| 27 | Spin 8000 g , 1degC           |                                 |
+----+-------------------------------+---------------------------------+
| 28 | Discard supernatant.          |                                 |
+----+-------------------------------+---------------------------------+
| 29 | Transfer pellet to 1.5mL      | Removes salts from DNA pellet.  |
|    | microfuge tube using 800 uL   |                                 |
|    | room temp 70% EtOH.           |                                 |
+----+-------------------------------+---------------------------------+
| 30 | Invert tube several times.    |                                 |
+----+-------------------------------+---------------------------------+
| 31 | Spin 5 min, 16 k\*g.          |                                 |
+----+-------------------------------+---------------------------------+
| 32 | Remove supernatant with       |                                 |
|    | pipette                       |                                 |
+----+-------------------------------+---------------------------------+
| 33 | Dry in Speed-Vac 5 min, high  |                                 |
|    | temp.                         |                                 |
+----+-------------------------------+---------------------------------+
| 34 | Dissolve pellet in 225 uL 10  |                                 |
|    | mM Tris pH 8.3                |                                 |
+----+-------------------------------+---------------------------------+
| *  |                               |                                 |
| Sp |                               |                                 |
| in |                               |                                 |
| Co |                               |                                 |
| lu |                               |                                 |
| mn |                               |                                 |
| P  |                               |                                 |
| ur |                               |                                 |
| if |                               |                                 |
| ic |                               |                                 |
| at |                               |                                 |
| io |                               |                                 |
| n* |                               |                                 |
+----+-------------------------------+---------------------------------+
| 35 | Add 450 uL (2 Volumes) MoBio  |                                 |
|    | S3, vortex 5s                 |                                 |
+----+-------------------------------+---------------------------------+
| 36 | Load to Mo Bio spin filter,   |                                 |
|    | spin 10k\*g, 1 min., discard  |                                 |
|    | flow-trough                   |                                 |
+----+-------------------------------+---------------------------------+
| 37 | Add 300 uL MoBio S4, spin     |                                 |
|    | 10k\*g 30 s., discard         |                                 |
|    | flow-throgh.                  |                                 |
+----+-------------------------------+---------------------------------+
| 38 | Spin an additional 1 min,     |                                 |
|    | 10k\*g.                       |                                 |
+----+-------------------------------+---------------------------------+
| 39 | Place spin filter in new      |                                 |
|    | tube.                         |                                 |
+----+-------------------------------+---------------------------------+
| 40 | Add 50 uL MoBio S5            |                                 |
+----+-------------------------------+---------------------------------+
| 41 | Spin 30 s 10k\*g.             |                                 |
+----+-------------------------------+---------------------------------+
|    |                               |                                 |
+----+-------------------------------+---------------------------------+

Linear Acrylamide (5000ug/mL stock) addition.

To achieve a final concentration of 15 ug/ul in the precipitation mix:

C­1 \* V1 = C2 \* V2

C1 \* V1 = C2 \* \[(Vaq + Vsa + V1) + 3(Vaq + Vsa + V1)\]

Where Vaq = Volume of aqueous solution containing nucleic acids of
interest

Vsa = Volume of 3M sodium acetate solution = 1/10 \* (Vaq + V1)

C1 \* V1 = C2 \* 4 \* (Vaq + Vsa + V1)

C1 \* V1 = C2 \* 4 \* (Vaq + (1/10 \* (Vaq + V1)) + V1)

C1 \* V1 = C2 \* 4 \* (Vaq + 0.1\*Vaq + 0.1\* V1 + V1)

C1 \* V1 = C2 \* 4 \* (1.1 \* Vaq + 1.1 \* V1)

V1 = 4 \* (C2/C1) \* (1.1) \* (Vaq + V1)

V1 = 4 \* (15 ug/mL / 5000 ug/mL) \* (1.1 \* Vaq + 1.1 \* V1)

V1 = 0.0132 \* Vaq + 0.0132 \* V1

0.9868\*V1 = 0.0132 \* Vaq

V1 = 0.0134\*Vaq

Therefore, add \~ 1.3% of Linear Acryamide

Example: If you have 200 uL DNA solution, add 2.6 uL of Linear
Acrylamide stock to achieve an ultimate concentration of \~ 15 ug/mL
considering the volumes of sodium acetate and ethanol.