When you have added chloroform and centrifuged, there will be separation of phases: Upper aqueous layer, Middle layer containing cell debris and lower organic layer containing chloroform. Phages will be present in the upper aqueous layer only. So, carefully separate the upper layer with micro pipette and avoid touching the middle layer which may contain traces of chloroform.

Dear Lee Jinsun,

Chloroform is usually used in phage preparations as this kills the bacterial cells used to produce phage by disrupting cell membranes, and PEG is usually used to aggregate phage particles into larger clumps to allow pelleting them by centrifugation.

Although Atif's suggestion should help you to prepare phage suspensions with minimal chloroform-contamination, you could try incubating your suspension with stirring at 37°C without a lid for 30 min to evaporate any remaining chloroform, or you could omit chloroform from your isolation protocol altogether and rely on low-speed centrifugation to remove cells from the culture supernatant before you add PEG to pellet the phage.

You could also consider modifying your titre assay. If chloroform contamination was reducing the titre of your phage preparations, you should be able to reduce this by either adding phage to a larger volume of lower-density host cells, pelleting the cells after infection and re-suspending in fresh buffer before plating out (i.e. effectively diluting the chloroform, though this also dilutes the phage), or by plating out phage directly onto plates, allowing them to dry (and the chloroform evaporate) before spreading the host cells (though this may also reduce the efficiency of infection).

Regards, Andrew.

Andrew J Spiers Thank you for your advices, I will applicate your protocol in experiments :) have a nice day

sincerely, Jinsun

Atif Khan thank you Khan, I will try as you advice, have a nice day :)

sincerely, Jinsun