Stealth鈩?nbsp;RNAi or siRNA Transfection
浠?/p>
24
瀛旀澘涓轰緥锛屽叾浣欒鏍肩殑杞煋瑙佽〃
1
1
涓澘锛岀粏鑳炲瘑搴︿负
30-50%
閫傚疁銆?/p>
娉ㄦ剰锛?/p>
鏍规嵁杞煋鍚庣粏鑳炴娴嬫椂闂撮暱鐭喅瀹氱粏鑳炰腑鏉垮瘑搴︼紝
濡傛灉杞煋鍚庨渶瑕侀暱鏃堕棿
鍚庢娴嬶紝鍒欑粏鑳炰腑鏉垮瘑搴﹂�傚綋闄嶄綆锛屽凡閬垮厤缁嗚優杩囧害鐢熼暱瀵艰嚧瀛樻椿闄嶄綆銆?/p>
2
绗簩澶╋紙
24-36
灏忔椂鍚庯級姣忎釜瀛旇浆鏌撴柟寮忓涓嬶細
A
灏?/p>
20pmol siRNA
婧朵簬
50ul Opti-mem
鏃犺娓呭煿鍏诲熀涓�?/p>
B
灏?/p>
1ul lipo2000
婧朵簬
50ul Opti-mem
鏃犺娓呭煿鍏诲熀涓紝娣峰寑瀹ゆ俯鏀剧疆
5min
銆?/p>
C
灏?/p>
A B
涓ょ娣峰悎锛屾斁缃?/p>
20min
銆?/p>
3
杞煋鏈熼棿锛屽皢
24
瀛旀澘鍩瑰吇鍩烘崲鎴愭棤琛�娓呭煿鍏诲熀锛屾瘡瀛?/p>
400ul
銆傚皢
C
绠?/p>
mix
鍔?/p>
鍏?/p>
24
瀛旀澘瀵瑰簲瀛斾腑锛?/p>
4-6
灏忔椂鍊欐崲鎴愭湁琛�娓呭煿鍏诲熀銆?/p>
Plasmid DNA Transfection
DNA(ug):lipo 2000(ul)=1:2-3
杞煋鏃剁粏鑳炲瘑搴﹁秺楂橈紝杞煋鏁堢巼锛岃〃杈炬晥鐜囦篃瓒婇珮锛屽苟涓斿彲浠ラ檷浣庣粏鑳炴瘨鎬с�?/p>
1
涓澘銆?/p>
璐村缁嗚優锛?/p>
0.5-2X10
5
cells/well
锛岀浜屽ぉ寰呯粏鑳炲瘑搴﹁揪鍒?/p>
90%
浠ヤ笂鏃惰浆鏌?/p>
鎮诞缁嗚優锛?/p>
4-8X10
5
cells/well
锛屼腑鏉垮悗闅忓嵆杞煋銆?/p>
2
杞煋銆?/p>
A
灏?/p>
0.8ug DNA
婧朵簬
50ul Opti-mem
鏃犺娓呭煿鍏诲熀涓�?/p>
B
灏?/p>
2ul lipo2000
婧朵簬
50ul Opti-mem
鏃犺娓呭煿鍏诲熀涓紝娣峰寑瀹ゆ俯鏀剧疆
5min
銆?/p>
C
灏?/p>
A B
涓ょ娣峰悎锛屾斁缃?/p>
20min
銆?/p>
杞煋鏈熼棿锛屽皢
24
瀛旀澘鍩瑰吇鍩烘崲鎴愭棤琛�娓呭煿鍏诲熀锛屾瘡瀛?/p>
400ul
銆傚皢
C
绠?/p>
mix
鍔犲叆
24
瀛旀澘瀵瑰簲瀛斾腑锛?/p>
4-6
灏忔椂鍊欐崲鎴愭湁琛�娓呭煿鍏诲熀銆?/p>