2.Ice15min. 3.Pelletcellsinappropriatecentrifugetube3-5000xg5min(~5000rpminaSorvallSS-34rotor) 4.Discardsupernatantandadd0.4volume(ieoforiginalvolume,hereitis40ml)TfbI,resupendandice15min. 5.Pelletcellsasin#3. 6.Discardsupernatantandresupendin0.04volumeTfbII,ice15minandeitheruseimmediatelyorquickfreezeat-70Cforstorage.Iusuallysavethesein0.25to0.5mlaliquots.Quickfreezeinethanol-dryiceorliquidnitrogenpriortostorageina-70to-80Cfreezer.Thawonicejustbeforeusinginatransformationexperiment. Itypicallytransform50ulcellswith2-10ulofaligationreaction,andyoushouldgetbetween1x10exp8to1x10exp9cfu"s/ugDNA.Procedure
1.Inoculate1mlfromovernightcultureinto100mlPsibroth(scaleupordownasneeded).Incubateat37CwithaerationtoA550=0.48MediumandBuffers
compound amount Bactoyeastextract 5g BactoTryptone 20g magnesiumsulfate 5g pH7.6withpotassiumhydroxide compound amount finalmolarity/conc. potassiumacetate .588g 30mM rubidiumchloride 2.42g 100mM calciumchloride 0.294g 10mM manganesechloride 2.0g 50mM glycerol 30ml 15%v/v pH5.8withdiluteaceticacid compound amount finalmolarity/conc. MOPS 0.21g 10mM calciumchloride 1.1g 75mM rubidiumchloride 0.121g 10mM glycerol 15ml 15%v/v pH6.5withdiluteNaOH
