Extraction of surface-community DNA from Ulva sp.
This method performs a direct lysis of the microbial community on the algal surface followed by an organic extraction and precipitation of the microbial DNA.
Ulva plants do get damaged during the treatment, however eukaryotic microbes might be lysed.
Therefore microscopic check of the algal surface before treatment and removal of basal parts (old and most likely covered by secondary colonisers) is necessary.
Collect several Ulva sp. plants by hand from intertidal pool and transfer into a plastic bag container with sea water (from the same site).
Transfer into the laboratory asap.
Transfer plants into sterile washing bowl container with a bit of CMFSW and cut off the buttom part (2-3 cm) of the plant with a sterile knife and forceps.
Chop the plant into piece of roughly 2-4 cm length width.
Wash plant pieces 3 times for 1-2 min with CMFSW; make sure smaller bits and non-plant material get wash away.
Take a couple of random pieces and perform a microscopic observation on it:place Ulva piece onto glass slidecover Ulva piece with approx. 10 µl of live stain.
Place on cover slip and drain excessive liquid with a tissue.
Observe under fluorescent microscope.
Describe and note surface coverage of cells.
Using a balance weigh out 12 g of plant wet-weight (drip dry) into a petri dish.
Transfer 12 g of plant into a sterile 50 ml Falcon tube with 30 ml of CMFSW and 300 µl of 3M Multi Enzyme Cleaner; shake gently to make sure the plant surface is covered with the CMFSW cleaner mix.
Incubate at room temperature.
Perform microscopy after 30 min (see point 6).
If surface shows little cells left or is cleared, then go to point 12.
Otherwise continue incubation (total 1 hour or 2 hours), until most cells on the plant surface are remove.
Transfer liquid into 1 or 2 new 50ml Falcon tubes (file tubes not more than 20 ml); make sure no plant material is being transferred, optional the liquid can be filtered through a sterile 125 µm sieve of a 0.8 µm filter.
Add an equal volume of phenol chloroform isoamyl alcohol (25:24:1) to the supernatant and shake gently but thoroughly.
Centrifuge for 5 min at 2000 x g.
Transfer aqueous phase into a SS34 tubes.
Add 1/10 volume of 3M sodium acetate and 3 volumes of 95% ethanol.
Shake gently but thoroughly; note: a white precipitate will become visible.
Incubate at –20oC for at least 2 hours.
Centrifuge for 30 min at 20 000 x g.
Remove supernatant.
Add 10 ml of 70% ethanol to pellet.
Centrifuge for 5 min at 20 000 x g.
Remove supernatant and air-dry pellet.
Resuspend pellet in 0.5 ml sterile water and transfer into 2 ml microcentrifuge tube.
Add 1/10 volume of 3M sodium acetate and 3 volumes of 95% ethanol.
Shake gently but thoroughly; note: a white precipitate will become visible.
Incubate at –20oC for at least 2 hours.
Centrifuge for 30 min at 20 000 x g.
Remove supernatant.
Add 1 ml of 70% ethanol to pellet.
Centrifuge for 5 min at 20 000 x g.
Remove supernatant and air-dry pellet.
Resuspend pellet into 50-100µl TE-buffer.
Check approx. 1µl on agarose gel.