1. 璇烽棶浣犱滑鐢ㄧ殑鏄摢绉嶈浆鏌撶殑鏂规硶? 鍖呰鐥呮瘨鏄敤鐨勫煿鍏荤毧鏄粈涔堣鏍肩殑锛

鎴戜滑鐢ㄧ殑鏄笁璐ㄧ矑绯荤粺鐨勭梾姣掑寘瑁呮柟娉曪紝鎵鐢ㄧ殑鍖呰鍣ㄧ毧涓150T鐨勭粏鑳炲煿鍏荤摱锛

 

2. 璇烽棶浣犱滑鐢ㄧ殑鍖呰璐ㄧ矑锛歱Helper1鍜宲Helper2鏄笉鏄氨鏄痑ddgene鐨刾MD2.G鍜宲sPAX2锛熸垜鐪嬪埌浣犱滑鐢ㄧ殑姣斾緥鏄5:1锛20ug鍜5ug锛夛紝浣犱滑鏄笉鏄祴璇曡繃涓嶅悓鐨勬瘮渚嬶紝鍙戠幇杩欎釜鏉′欢鏈濂藉憿锛

鎴戜滑鐢ㄧ殑鍖呰璐ㄧ矑鏄痯R8.74鍜宲VSV-G锛岀梾姣掑寘瑁呮柟娉曟槸鍏徃鐥呮瘨鍖呰骞冲彴鐨勫悓浜嬪仛杩囦紭鍖栫殑锛

 

3. 浣犱滑鐨勬枃搴撶梾姣掔矘搴﹀緢澶э紝鏄笉鏄祿缂╄繃绋嬩腑鍩瑰吇鍩轰腑鐨勮娓呬篃琚祿缂╀簡鍛紵

姝e儚鎮ㄦ墍璇寸殑锛屾枃搴撶梾姣掔矘搴︽瘮杈冨ぇ鏄洜涓哄湪鐥呮瘨娴撶缉杩囩▼涓紝鍩瑰吇鍩轰腑鐨勬垚鍒嗕篃琚祿缂╀簡锛

 

4. 杞煋璇曞墏鐢ㄧ殑鏄粈涔堬紵

杞煋璇曞墏锛歅EI锛

 

5. PCR娴嬪簭鏈変粈涔堥渶瑕佹敞鎰忕殑鍦版柟鍚楋紵

PCR娌′粈涔堣娉ㄦ剰鐨勶紝鍙淇濊瘉鏀堕泦鐨勭粏鑳炴暟鑳藉瑕嗙洊鏁翠釜鏂囧簱灏卞彲浠ワ紝 鐒跺悗杩欎簺缁嗚優鎻愬嚭鏉ョ殑鍩哄洜缁勫叏閮ㄧ敤鏉ユ墿澧濸CR锛 寰幆鏁颁笉鑳藉お澶 20-25涓惊鐜紱

 

6. 鏂囧簱鐥呮瘨鐨勬淮搴﹀ソ鍍忔瘮璇存槑涔﹂噷闈㈡彁渚涚殑婊村害瑕侀珮寰堝锛屾垜鏄敤鐨293T缁嗚優锛屽ぇ鑷存祴涓涓嬫劅鏌撶殑鏁堢巼锛屽彂鐜1:10000绋閲婁箣鍚庣敤200ul鍩瑰吇鍩猴紝鎰熸煋涔嬪悗锛宲uro鍩烘湰鏉涓嶆浠讳綍缁嗚優锛屽鐓ф湭鎰熸煋鐨勭粏鑳炲叏閮ㄦ潃姝伙紝鐥呮瘨鐨勬淮搴﹁繙姣斾綘浠彁渚涚殑鏁板奸珮锛

铏界劧缁嗚優鏍竴鏍凤紝浣嗘槸缁嗚優鐘舵佷笉涓鏍风殑璇濓紝婊村害涔熻繕鏄細鏈夊樊鍒殑锛涙墍浠ユ垜浠細寤鸿鍦ㄦ嬁鍒扮梾姣掍箣鍚庯紝鍦ㄩ澏缁嗚優涓婇噸鏂版祴瀹氱梾姣掓淮搴︼紝鍐冲畾鐥呮瘨鐢ㄩ噺

 

7. 绛涢夊嚭闃虫х殑缁嗚優涔嬪悗锛岃鎻愬熀鍥犵粍杩涜PCR锛屽彲浠ユ彁渚汸CR寮曠墿鍚楋紵

Library-Seq-F1: N25TATCTTGTGGAAAGGACGAAACACC
Library-Seq-R锛歂25AATACGGTTATCCACGCGGC

 

8.浣犱滑鐨勬參鐥呮瘨鏄庝箞娴撶缉鐨勶紵

鐢ㄨ秴婊ょ娴撶缉鐨勶紱