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產品說明:
RNAi library includes about 150,000 lentivirus-based shRNA reagents targeting 16,000 genes of human and mouse respectively. The shRNA constructs, which are based on pLKO.1 backbone (with ampicillin selection marker) that can support production of VSV-G-peudotyped shRNA-expressing lentivirus by co-transfection with pCMVΔR8.91 and pMD.G, were transformed to DH5a bacterium. RNAi core provides the bacterial stock containing the shRNA construct. Lentivirus-based shRNAs transfer vector are used for gene silencing either by transfecting the shRNA construct directly or by transducing the shRNA lentivirus into cells. Stable cell lines could be established by puromycin selection after transduction.

使用需知:


  1.  所有訂購之細菌株均為96 well plate format。

  2.  細菌株均為15% glycerol stock,體積約為100μl / well。

  3.  處理程序:回溫→離心(2000rpm, 1min)→撕封膜。

  4.   請以streak out 的方式挑選單一菌落,經16小時培養之後,單一菌落直徑約為0.5mm大小, 請勿挑選培養盤上菌落較大者作液體培養,以防選到變異株; 每一clone至少挑選2顆菌落,之後萃取質體,並做定序 (註一)。 若塗佈養菌失敗,可先從stock進行少量液體培養後,再以streak out方式挑菌。

 5.   本細菌庫所使用之質體為pLKO.1(Amp+),生長速度較慢,故建議液體培養時請使用Terrific Broth (TB,註二)。

*註一: pLKO.1-puro轉移載體以U6 promoter表達shRNA,本核心設施使用下之primers定序之:
(1) LKO_shRNA/F (forward primer):
5’-acaaaatacgtgacgtag-3’ (for sequencing forward strand of shRNA)
(2) LKO_shRNA/R (reverse primer):
5’-ctgttgctattatgtctac-3’ (for sequencing reverse strand of shRNA)
shRNA因有二級結構,本核心設施建議定序方法如下:
(1) 請在樣品中加入5% ~10% DMSO,定序之;或
(2) 請定序中心以dGTP BigDye V1.1 kit或類似產品定序之。

二級結構有解開(圖片按下去可放大) 二級結構沒解開(圖片按下去可放大)

*註二: Terrific Broth 配方,正確配法請參考 Molecular cloning。

 Tryptone 12g/L
 Yeast extract 24g/L
 Glycerol(100%) 4ml/L
 KH2PO4 2.31g/L
 K2HPO4 12.54g/L

Ampicillin or Carbenicillin final concentration:100mg / L

# 當您的實驗室開始使用細菌株進行實驗後,如果有【定序】、【Knockdown結果】或【論文發表】時,請feedback至本核心設施,俾使整合shRNA database或將成果(論文)呈報到MOST,謝謝合作 #  

技術支援:


  1.   shRNA定序引子 (primer sequence for shRNA sequencing),請參閱【常見問答集之shRNA相關-Q2

*註三: 領取菌株時,請務必攜帶保麗龍盒及乾冰,如有需要訂購保麗龍盒及乾冰,請事先聯絡鍾小姐(02)2789-9856#15。

 
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