Supplies

For extraction of 1 litre of induced Bradyrhizobium japonicum culture requires:

40    grams               XAD-2 Resin (Alltech, XAD-1180 20/60 mesh)

20 ml                      methanol (added to culture)

40 ml                      methanol (elution solvent from resin)

30 ml                      acetone (elution solvent from resin)

2 ml                        18% acetonitrile solution

1                                                        funnel fitted with brass screen

1                                                        2 litre flask

1                                                        250 ml boiling flask

1                            47 mm Whatman #1 filter paper circle

Equipment

Shaker

Flow Cabinet

Fume Hood

Rotary Evaporator

Thermolyne Tube Vortexer

2-litre vacuum filtration system with fritted glass base

Extraction Procedure

1.      Prepare a 1-litre Bradyrhizobium japonicum culture that has been induced with 5 µM of genistein. Grow culture to OD600 of approximately 0.2 to 0.4 .

2.      Before using XAD resins, they must be first conditioned. Place resin in the funnel of the vacuum filtration unit. For each 40 grams of XAD resin, rinse with two washings of 20 ml of acetone, followed by conditioning with 20 ml of methanol, and finally two washings of water (20 ml). Do not use vacuum during each wash. Wash for 1 minute, and then apply vacuum to remove the solvents or water into the collection flask. Allow all water to be removed.

3.      To the culture, add 2% of methanol; for a 1-litre culture use 20 ml of methanol.

4.      Weigh 40 grams of resin and add to flask containing the culture; note that you do not have to centrifuge culture to remove cell pellet.

5.      Place culture flask and resin on shaker and shake overnight (at least 6 hours) at 150 rpm.

6.      After shaking culture and resin, separate culture and resin by pouring the culture through a funnel fitted with a coarse brass mesh. The mesh should be fine enough to allow the culture to flow quickly through, but will filter out the resin beads without any loss. You can fit the funnel over a 2-litre flask to collect the culture. Use water to wash remaining resin beads into funnel from culture flask, and then wash the resin beads that you have collected in the funnel with 1 litre of water.

7.      Fit the fritted glass base of vacuum filtration system with the Whatman #1 paper disk. The purpose of the disk is to collect the beads and to keep  the solvents with the beads until the vacuum is applied. Only when the vacuum is applied should the solvents pass through the glass frit base into the collection flask.

8.      Transfer the resin beads to the funnel on the vacuum filtration system. Use a water bottle to wash all the resin beads into the filter funnel. Apply vacuum to remove water from beads. Discard water collected in flask and rinse flask with methanol to remove water traces.

9.      Wash beads with 40 ml of methanol. Let the methanol wash the beads for 1 minute before applying vacuum. Apply vacuum to system, and collect methanol in flask. After all methanol has been collected, remove vacuum from system.

10.   Wash beads with 30 ml of acetone. As with methanol, wash the beads with the acetone for 1 minute, after which you can apply vacuum to pull the acetone through the filter paper and glass base into the flask. At this time both the methanol and the acetone filtrate are in the flask.

11.  Transfer filtrate to a 250 ml boiling flask and place on a rotary evaporator with a water bath temperature of 45°C and a speed of 125 rpm. Evaporate solvents until flask is dry.

12.  Add 2 ml of 18% acetonitrile to flask and wash the walls of the flask using a vortexer to ensure all residues are dissolved in the solution.

13.  Collect the solution and place in micro centrifuge tube, and spin at 10,000 rpm to sediment particulates. Label and store at -20°C until further analysis by HPLC.

HPLC Analysis

We  have analysed the Nod Factor extract using a Waters HPLC system consisting of the following components;

i) 2 model 510 HPLC pumps

ii) Waters 710 WISP automatic sample injector

iii)Vydac 5µM 300 A c18 column 218TP54  4.6 x 250 mm using a column pre-filter

iv) Waters 410 UV detector at 210 nm

Flow rate: 1.0 ml/min

Solvent: Initial 18:82 acetonitrile:water  gradient as follows;

10-30 minutes 18% to 60% acetonitrile

30-35 minutes 60% to 100% acetonitrile

35-40 minutes 100% to 18% acetonitrile

40-45 minutes 18% acetonitrile

Run Time: 45 minutes

Nominal Elution Time for Nod Factor:  30.75-30.95 minutes

Retention time is consistent with Nod Factor standards that were injected

50 µl of samples are injected.