Life Technologies Product: Cat. No.: 18324-012 Lot No.: Size: 1 ml, 2 mg/ml Storage Conditions: 4 C DO NOT FREEZE MIX GENTLY BEFORE USE hPoFEt~rAMlNE Re~gent is ~ 3:1 (w/w) liposome formultuion of the polyct~tionic lipid 2.3-diokylo~iy-N-12(spermineciubout midokd~yll-N,N-dimethyl. I-prop~minium miluon~ cottue (DOSP ) (Chemic31 Abstr,tcts Regis~ty n3mo: N-l2-((2,5-blsl(3- minopropyl)3nuno~ o~ypentyl)lutuno) thy1]-N,N dimerhyl-2,3-bis(9-oct docenylo~iy)- I -prop3n mitdum u ifluoro~tcett~Le)~ nd rhe neutr31 lipid dioleoyl phospht~tidylethnol3ntu~e (DOPE) m mombrnefllteredw-ter. Itis~uitbleforthotttnsfectionofDNAuntocullu~8euhuyoticcell~(1). OnemiDiliteris~ufficientfot50to200triulsfection~ on 35-mm tirsue culture dishe~t or 15 to 70 tr nsfections on oO-mm dishes. Ilt~Ft llltF FOR TRANCt~NT f)R STARLE ~1ll: [A di~gr m of Lhis procedure ~tppe3rs in hgure 11. The rioncentr~dons of DNA 3nd Ltl~oF~crAMlNE Re~tgent, cell density ~nd triulsfectiom omes in these tnmsfecDon protocols were delerrnm~d wlrh me l3~mids pSV2-CAT nd pCMVCAT in 13HK-21, H-Li~ C05-7, CHO KI, NIH3T3, PC12 3nd Jurht ceD lines s well rs p3ss~ged pnmrry humrn f;brobl sts (1). SL3bb rr3nsfortn dons wete perfortned with pRSVn~o pl smid DNA m NIH3T3 cells (1). These condiuons 3re recommeno d .c uidelines only Whib 35-mm w~Ds or dishes re r doo,u 1~ for most ~pplict~tions, I rget sh~ed vessel~ re ~omeDmes required. For l rger DriSUe cuDure dishe~ T3ble I shows tho ~uggesu~d 3mounu of n 3~enu. Adjwt iDhmoumu in propotdon to Lhe ch~tnge in culture dD~h ~urf3ce 3re3. 1. in ~ sbuw~ll or 35-mm dssuo culture pliue, seed -1-3 L 101 eells p r weU un 2 ml of tto 3ppr0prLIlte compbue gruwth merdum (widh semm if cells 3re nornuUy cultured h the pre ence of serum). 2. Incubt~temeceDs~t37-Cin~COtmcubtuorundlthec~11s3ro50.80%confluem. ThiswdlwutJlyt31rel8-24hours,bulthedmewillv3rytmong cell types. (Opdmtl cell density m y vt ty widh ceU typo or ~pplie don. Since u! nsfecdon ~ffQ~ncy is senridv~ lo culture confluenceul ls imporuu~llomtmuunsttnd rd~eedin~pmtocolftom~iLpertmentto~ipenment) 3. preptue d~o followung ~oludon~ h 12- Ji 75-mm ~uonk Dlbct: Soludon A: For e3ch trtwfecdon, d31uue 1-2 LL8 Of DNA muo 100 ~1 erum-free me~3um. OPn-MEM0 I Reduced S~rum Medium (GIRCO BRL C3t. No. 31985) dve~ opdm31 resulU. Soludon B- For ~3ch ulwfection, diluue 2-25 ,LLI of LLDoFEaAMlNE Re3g~nt into 100 ,LLI rerum-free medium. 4. Combme d;e rwo soludons, mbl pndy, 3nd incub Le 3t room emper nue for 1545 mdn to tllow DNA-liposome compk~i~s Lo funm. The ~oluOon m3y ~ppe3r cloudy, 31dbough thi~ wfll not impede dle triwfecdon. Wh3b compiQies fonn, rinse dle cell~ once widh 2 ml of serum fn~e medium. 5. For e ch utwfecdon, il8d 0.8 ml of serum-free me~oum uo me mbo conulining the comple~u. Mik g~ntdy 3nd overl-y me doluted compleu soluoon ontothennsedeell~. Meoumconttudngserumm3ybetddedtodtecompb~Lui~ttt,w~uep(~oeNoue3). Donot3dd3ndb3ctenilhlgenutorned~ during u nsf~don. 6. Incub3te me eehs with tho compknet for 2-24 hour~ td 3rc in CO~ incubt,toL GIBCO BRL recommends ~uudng wito 5 hourt . 7. FoDowin~ incub don, 3dd I m3 of growmh medium conuur3ng twice tho nornuLI concenu~don of serum withoul removung Lhe 03nsfecOon mu~cnue. (If serum wils included in ~uep 5, 3dd I mi of cnmpleuo growth medium u thi~ dme.) If touicity u ~ problem, nemov~ Lhe u 3nsfecdon m3~Lnure nd nepbce il with compbte growot metoum. 8. R~pbce the med3um with Ituh eompleto met,8um U 18-24 houL~ following the ~ort of u r4fecdon. 9 Assi~y cell e~itri~cU for g~ne tcdvity 24-72 hours 3fter toe ~urt of u nsfecdon, depending on ceD type i~nd promoter 3cdvity. 10. A~imil3uprocedunecnbowedtotnuufectDNAfor~t3bkeuptes~ion. At72hours3fterO3nsfecdon,p3ssitg~mecell~1:10intoLher~elecnv~ medium for the reporter pne utwfeclaL For wuutce, for pRSVn~o u~n fecdons, medium ~hould cont3in GtiNErtaN0 ~GIBCO BRL Ctit. No. 11811). PRoCEDURE FOR 'rRANSlENT TRANSFFCTION OF SUSPFNSION CRU C: Wuh th~ celi,t once with serum-fieo growth meti3um withoul i~ndb~tcteny t~g~nu. 2 In ~ ~iri-well or 35-mm drisuo culnure plt~te, seed cell~ Y ~ densiq of 2-3 ri 10~ per well in 0.8 ml ~froe growLh medium. 3. preptue the followin~ oludon~ in 12- iL 75-mm ~uenb nube~i: Soludon A: For e3ch o nsfodon doluuo 1-3 llg of DNA muo 100 ,LD serum-freo medoum (ot~n-MEM0 I Reduced Serum Medium giv~ opdm31 re ults) Soludon B: FOT ~i~ch o nsf~cdPn doluu 2-25 ,_1 of LtFoFEcTAMlNE Ret~gent inuP 10~3 ,ul of senum-fr~e mediuriL 4. Combine drc two sPbdons~ mia Fndy imd ulcubt~ue t~t room temperrttuue 15-45 min. to t8bw DNA-liposome compleae~ to form. 5. Add the compbau to tht~ ce_ ~wpenrriont, mia gendy to en~uTe uniform di~o ibudon, t,nd incubue 2-24 hours u 37-C in CO~ incubtrLor. GIBCO BRL n cPmmends ~tiudng whlt 5 hour~. 6. FoDowing incubt~don, t~d 4 ml nf the trpprnprittt2 compbte gTowth medium (with serum) to e clt well. Incub~ue celi~ i~t 37-C in CO~ incubrLtri~T for ~ tottJ of 24-72 htnu2t. CoDect cells by ctin~rifuguion imd ~y cell eau~cu for Fne rtcdvity. |