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<p>Midi-prep &Transfection</p>
<p> </p>
<p> </p>
<div>1.Opti-MEM+ DNA(each: 3ug)= 250ul</div>
<div>2.(Opti-MEM 241ul+TR 9ul)*2 master mix (vortex well)</div>
<div>3.Put 2 into 1 250ul per each & mix(1min) , stand 30min</div>
<div>4.Treat #3 to H-293 Cell, media change after 10hours</div>
<div>5.Change media to serum free (starvation) Media change after 1day (Check GFP signal)</div>
<div>6.Prepare lysate after 2days(between 24~48hours) (check whether plasmid is well overexpressed by western blot)</div>
<div>7.Put Media 520ul per each in e.tube and 4 celcius 13000rpm, 1min centrifuge, get 500ul of each centrifuged into column and do 45min 13000rpm, 4celcius centrifuge</div>
<p>(Media = conditioned media(CM))</p>
<p>* Store rest of media는 in other tube (labeling)</p>
<p>8. Wash rest of cell in 1X PBS(cold) and after wash, do suction</p>
<p>9. Treat Lysis buffer 200ul and scrape using stripper. Collect all of them in E.tube & labeling, incubation 10 min in cold ice, then 15min 13000rpm, 4celcius centrifuge</p>
<p>(After sup media of centrifuged = cell extract(TCE))</p>
<p> </p>
<p>Transfection</p>
<p> </p>
<p> </p>
<div>1.Opti-MEM+ DNA(each: 3ug)= 250ul</div>
<div>2.(Opti-MEM 241ul+TR 9ul)*2 master mix (vortex well)</div>
<div>3.Put 2 into 1 250ul per each & mix(1min) , stand 30min</div>
<div>4.Treat #3 to H-293 Cell, media change after 10hours</div>
<div>5.Change media to serum free (starvation) Media change after 1day (Check GFP signal)</div>
<div>6.Prepare lysate after 2days(between 24~48hours) (check whether plasmid is well overexpressed by western blot)</div>
<div>7.Put Media 520ul per each in e.tube and 4 celcius 13000rpm, 1min centrifuge, get 500ul of each centrifuged into column and do 45min 13000rpm, 4celcius centrifuge</div>
<p>(Media = conditioned media(CM))</p>
<p>* Store rest of media는 in other tube (labeling)</p>
<p>8. Wash rest of cell in 1X PBS(cold) and after wash, do suction</p>
<p>9. Treat Lysis buffer 200ul and scrape using stripper. Collect all of them in E.tube & labeling, incubation 10 min in cold ice, then 15min 13000rpm, 4celcius centrifuge</p>
<p>(After sup media of centrifuged = cell extract(TCE))</p>
<p> </p>