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                                                                                               普通siRNA合成
產品簡介

普通的siRNA oligo均由HPLC純化,100%除去尚未配對的單鏈,價格便宜,質量上乘。研究者只需用包裝盒內的通用緩沖溶液稀釋后即可用e轉染試劑進行后續實驗。

實驗方法

siRNA 轉染  
A.siRNA轉染的方法  
  脂質體型轉染試劑進行轉染需要注重的幾點
哺乳動物轉染的常見方法有:磷酸鈣共沉淀、電穿孔法、DEAE-葡聚糖和polybrene、機械法(例如,顯微注射和基因槍)、陽離子脂質體試劑,其中陽離子脂質體試劑轉染法是目前最常用的轉染方法。
  • 轉染試劑的用量
  • siRNA的用量 
  • 轉染時的細胞密度
  • 轉染時的操作順序 
  • 轉染時的操作順序 
 
  • 細胞與轉染試劑/siRNA復合物的溫浴的時間
B.Lipofectamin2000 轉染試劑  
  Lipofectamin2000的應用領域
選擇最適合的轉染試劑和轉染條件,往往取決于不同的哺乳動物細胞類型和不同的核酸分子。 Lipofectamin2000適用于核酸的體內和體外操作,可應用于DNA、RNA、反義寡核苷酸、siRNA的轉染,也可應用于DNA/siRNA 的共轉染操作;是一種新型的高效siRNA轉染試劑。
  • 原代培養細胞和轉化細胞株的基因轉染
 
 
  • siRNA高通量轉染試驗
 
 
 
  • DNA轉染;DNA和siRNA的共轉染
 
 
 
  • 核酸(siRNA、DNA、RNA)的體內導入試驗
 
 
 
  • 貼壁細胞和懸浮細胞轉染
   
  Lipofectamin2000 的特點:
  不必更換培養基,操作簡便易行,可在半小時內完成操作
  在含血清培養基中也能表現高轉染效率
  細胞毒性低;適用細胞廣泛
  即用型試劑,可在含有抗生素的完全培養基中轉染
  基于脂質的轉染試劑,確保沒有RNAse活性
  □ 可介導siRNA高轉染細胞及體內siRNA的高效導入
C.Lipofectamin2000適用的細胞類型   
Lipofectamin2000轉染試劑可廣泛應用于多種細胞系的DNA和siRNA轉染如:

HeLa(人頸部癌 細胞)、MCF-7(人乳房癌細胞)、Hep3B(人肝細胞癌細胞)、COS-7(猴腎細胞)、Neuro-2a(鼠神經母細胞瘤細胞)、NIKS(人角 質化細胞)、B16(鼠黑素瘤細胞)、DLD-1(人結腸癌細胞)、NIH/3T3(鼠胚胎成纖維細胞)、HT-29(人結腸腺癌細胞)、A549(人肺 癌細胞)、CHO-k1(倉鼠卵巢細胞)和293(腺病毒5 DNA轉化的人胚胎腎細胞),SVRbag4細胞等。
       


D.轉染前細胞培養  
  在細胞板上培養細胞時,應使細胞匯合在24小時內達到70-90%。
  細胞培養用品 表面積(mm2/孔) 細胞密度 培養基(μL /孔)  
  96孔板 50 1.5´104-5.0´104 100μL  
  48孔板 100 3.0´104-1.0´105 200μL  
  24孔板 200 8.0´104-2.0´105 500μL  
  12孔板 401 1.6´105-4.0´105 1.0 mL  
  6孔板 962 3.0´105-8.0´105 2.0 mL  
  35 mm 962 3.0´105-8.0´105 2.0 mL  
  60 mm 2827 1.0´106-2.5´106 6.0 mL  
E.合適的lipofectamin2000用量  
  合適的siRNA(DNA):lipofetamin2000比例對核酸的高效轉染有重要影響;我們推薦的 DNA:lipofetamin2000為1:0.5—1:5(ug:ul),siRNA:lipofectamin2000為 1:0.01-1:0.1(pmol:ul)一般情況下,此范圍內都可獲得高的轉染效率。
細胞培養用品 siRNA/DNA 培養基最終體積 Lipofectamin2000(siRNA/DNA)  
96孔板 5pmol/0.2μg 100μL 0.25μl/0.5μl  
24孔板 20pmol/0.8μg 500μL 1μl /2μl  
12孔板 40 pmol /1.6μg 1 mL 2μl /4μl  
6孔板 100 pmol /4.0μg 2 mL 5μl /10μl  
35 mm 100 pmol /4.0μg 2 mL 5μl /10μl  
60 mm 600 pmol /8.0μg 5 mL 10μl /20μl  
F.貼壁細胞轉染程序     
  轉染前一天,4-5´104細胞接種在24孔板上, 0.5mL含FBS和抗生素的DMEM(或Opti-MEM,其他培養基)細胞培養基。  
 
  
選用生理狀態良好的細胞對提高轉染效率很 2. 選擇用于初期接種的細胞數量,應能在24小時內使細胞匯合達到70-90%。  
重要。siRNA(DNA)和lipofectamin的用量和兩者的比例可在推薦范圍內適當調整。  
  在50μl的DMEM(或Opti-MEM,或其他無血清培養基)無血清培養基加入20pmol siRNA(或0.8μg DNA),柔和混勻;  
   
    
  混勻lipofectamin試劑,用50μl無血清的DMEM或Opti-MEM,或其他無血清培養基)稀釋1μl lipofectamin試劑(DNA轉染時,則加入2μllipofectamin試劑),輕輕混勻,室溫放置5分鐘;  
 
  
  將稀釋好的siRNA和RNAi-Mate試劑混合;輕柔混勻,室溫放置20分鐘,以便形成siRNA/lipofectamin(或DNA/lipofectamin)復合物。  
 
  
  將100μl siRNA/lipofectamin(或DNA/lipofectamin)復合物加到含有細胞和培養基的培養板的孔中,來回輕柔搖晃細胞培養板板。  
 
  
  7. 細胞在CO2培養箱中37℃溫育24h-48h后,進行轉染后的其它檢測步驟。如果細胞株比較敏感,孵育4-6小時后,除去復合物,更換培養基。    
G.懸浮細胞轉染程序     
  轉染的當天,收集細胞離心,用含FBS的培養基重懸。  
 
  
  在50μl的DMEM(或Opti-MEM,或其他無血清培養基)無血清的培養基加入20pmol siRNA(或0.8μg DNA),柔和混勻;  
 
  
  混勻lipofectamin試劑,用50μl無血清的DMEM或Opti-MEM,或其他無血清培養基)稀釋1μl lipofectamin試劑(DNA轉染時,則加入2μl lipofectamin試劑),輕輕混勻,室溫放置5分鐘;  
 
  
  將稀釋好的siRNA和lipofectamin試劑混合;輕柔混勻,室溫放置20分鐘,以便形成siRNA/lipofectamin(或DNA/lipofectamin)復合物。  
 
  
  再加入400μL細胞懸浮液(細胞數量決定于細胞類型和轉染后分析測試的時間)。  
 
  
  6. 細胞在CO2培養箱中37℃溫育24h-48h后,進行轉染后的其它檢測步驟。如果細胞株比較敏感,孵育4-6小時后,除去復合物,更換培養基。    
     
     
     
     
     
H.DNA和siRNA共轉染細胞  
  在轉染的前一天,4-5´104細胞接種在24孔板上,0.5 mL含FBS和抗生素的細胞培養基。
  選擇用于初期接種的細胞密度,應能在24小時內使細胞匯合達到70-90%。
  在100μL的無血清的培養基中稀釋20pmol siRNA和0.2μg DNA,加入2μl lipofectamin試劑,充分混合,放置20分鐘,以便形成siRNA/ DNA/lipofectamin復合物。
  將siRNA/ DNA/lipofectamin復合物加入培養基中,輕輕混勻。
  5. 細胞在37℃溫育24h-48h后,進行轉染后的其它步驟。
I.siRNA體內導入方法  
  適量的siRNA或DNA溶于不含RNA酶的無菌水中,輕輕混勻,因為注射液體積有限,建議采用高濃度的siRNA或DNA,一般DNA為2μg /μL、siRNA為10μg /μL。
  取適量的DNA、siRNA或siRNA\DNA復合物與lipofectamin混合。例如,在1#管中加入0.5μL的DNA(1μg)和 0.5μL的siRNA(5μg),在2#管中加入0.55μL的lipofectamin(24μg)和0.45μL不含RNA酶的無菌水中,將1#管 中的溶液加入2#管中,在室溫下溫育30分鐘,以形成siRNA/DNA-lipofectamin復合物。
  3. 制備的siRNA/DNA-lipofectamine復合物可用于體內導入siRNA、DNA或siRNA\DNA。

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