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 .
