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请问northern blot杂交液只用7% SDS in 0.25 M sodium phosphate buffer pH 7.2行吗
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大家好 我看有些文献上做siRNA BLOT 只用7% SDS in 0.25 M sodium phosphate buffer pH 7.2 请问有人做过吗,谢谢 如: Northern blot analyses Total RNA (30–50 g) was separated on a 15% denaturing polyacrylamide Tris/borate/EDTA gel; the gel was then soaked in 10 mM sodium phosphate buffer pH 7.0 and subsequently in 20 SSC for 10 min. The RNAs were transferred by overnight capillary blotting in 20 SSC to Zeta-probe (Bio-Rad) or Hybond N+ (Amersham Biosciences) membranes. Membranes were crosslinked and/or baked for 2 h at 80 °C. Probes complementary to sRNAs were end-labelled with [ -32P]ATP with the use of T4 polynucleotide kinase (New England BioLabs). Hybridization was performed in 5 ml of 7% SDS in 0.25 M sodium phosphate buffer pH 7.2, and the hybridization temperature was set 15 °C below the Tm of oligonucleotides. Membranes were washed twice at hybridization temperature with 2 SSC, 0.1% SDS for 10 min, and exposed to PhosphorImage plates. Decade RNA marker was labelled in accordance with the manufacturer's instruction (Ambion). |
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