HP-20 resin Ethyl Acetate (EtOAc) extractions from liquid cultures
HP-20 resin and Media Preparation
HP-20 resin activation: Cover 100 g of dried HP-20 resin with approximately 300 mL of EtOAc in a clean glass bottle with polypropylene lid and shake for 10 minutes. Filter the HP-20 resin using a Buchnar funnel and filter paper and discard the EtOAc into an appropriate flammable solvent waste container. Add HP-20 resin back into the used bottle and cover again with 300 mL ethyl acetate.
Adding HP-20 resin to media: Dispense 50 mL of media into 250 mL conical flasks. Use a Buchner funnel and filter paper to weigh out 5% w/v of HP-20 resin (2.5 g of HP-20 resin per 50 mL of media). Once completely dry (no resin clumping), add 2.5 g oh HP.20 resin per 50 mL of media and autoclave.
Notes: the HP-20 resin should be completely dry. Never contact plastic with EtOAc or HP-20 resin that has been stored in EtOAc. Add HP-20 resin to fermentation media prior to autoclaving.
Bacterial cultures in HP-20 resin and metabolite extraction
Add 2.5 mL of a bacterial pre-culture into the conical flask containing 50 mL of media and 5% of HP-20. Incubate the culture for the required time in a shaker. After incubation, transfer the culture from the conical flask into a 50 mL Falcon tube. Centrifuge for 10 mins at 4°C and 5,000 rpm. Remove the tube from the centrifuge, discard the supernatant and freeze at - 80°C the cell pellet/HP-20 resin overnight. After frozen, place the tube in a freeze-dryer until completely dry (no clumps / humidity should be observed). Transfer the dry cell pellet/HP-20 resin to a clean Erlenmeyer flask (50 mL). Add 20 mL of EtOAc and shake overnight (100 rpm). Filter the EtOAc into a pre-weighted 20 mL glass scintillation vial with filter paper. Again, add 20 mL of EtOAc and do the same process as before. Evaporate EtOAc each time under nitrogen and record the extract weight.
Notes: for scale-up experiments, scale-up the bacterial cultures and add the required amount of HP-20 resin and EtOAc.
Ethyl Acetate (EtOAc) extractions from solid media cultures
For bacterial cultures from solid media, transfer the wished cultures to a Falcon tubes (50 mL). Freeze-dry samples as previously explained. Transfer sample into a clean Erlenmeyer flask (50 mL) and extract metabolites twice with 20 mL EtOAc. Remove EtOAc each time into a pre-weighted 20 mL glass scintillation vial. Evaporate under nitrogen and record the extract weight.
Notes: for scale-up experiments, scale-up the bacterial cultures and add the required amount of EtOAc.