Sampling for RNA / Protein / dissolved nutrients from phage infection experiments
At each time point , collect 10ml from each experimental bottle / tube into a 15ml conical tube , using a serological pipet .
Collect one 10ml sample for RNA and one 10ml sample for protein .
Spin tubes for 15 minutes at 4700 rpm ( 4816 x g ) in a swinging bucket rotor .
If you wish to collect the cell - free fraction ( e . g .
for analyzing dissolved inorganic or organic nutrients or phage particles ) : decant supernatants into labeled sterile 50 ml conical tubes .
We pool the RNA and protein supernatants together resulting in a 20ml combined supernatant .
If you do not wish to collect the cell - free fraction : decant supernatant into waste container .
Flash - freeze cell pellets in 15 ml tubes using liquid nitrogen ; store pellets in - 80°C freezer until later analysis .
If you are not collecting the cell - free fraction , stop here .
If you are collecting the cell - free fraction , go to Step 5 .
Remove the plunger from a 20ml clean syringe .
Attach a new 0.2µm syringe filter to the syringe .
Pour your 20ml combined supernatant into the barrel of the syringe .
Replace plunger on the syringe and filter your 20ml supernatant into appropriate tubes for your downstream nutrient / phage analysis ( we use a 15ml conical tube plus a 5ml snap - cap Eppendorf tube ) .
Repeat step 5 for each sample .
You can reuse the same syringe for replicates of the same condition / treatment .
Store 0.2µm filtered samples in - 20ºC until analysis .
( if analyzing phage particles in the filtrate , store at 4ºC instead . )
