SPLITTING BHKs分裂BHKS

SPLITTING BHKs Reagents α MEM medium for BHK :α MEM Invitrogen cata # 1256110% Hyclone Heat inact

SPLITTING BHKs Reagents αMEM medium for BHK : αMEM Invitrogen cata #12561 10% Hyclone Heat inactivated FCS 1% HEPES Invitrogen 1%P/S Invitrogen Other reagents HBSS :Invitogen 14170 LMP Agar :Fisher :catalogue #B__ 50100 2X MEM :Invitrogen catalogue #11935046 When the BHK are confluent, they need to be split (They will appear “stringy” if they are overly confluent .You will need to thaw and start over if this happens) Draw all the media out the flask .Add 5ml of HBSS (With 1% HEPES 5Mm EDTA )to the flask .Incubate 37 C5-10 min .(Less time is better the __lls don’t like EDTA) Following the incubation, gently “bang “the __lls off of the flask, being sure to wash the bottom of the flask, being sure that the EDTA is rinsed off. Draw up 12 ml of media in the flask into 50 ml conical and spin 3min at 1200 rpm 4C. On__ the __lls h__e finished spinning, bringing up to 10 ml of medium and split according your needs .If you need the flask confluent next day dilute 1: 10 and 2days later 1: 20. They grow fast .