Northern Blot of tRNA in Yeast
酵母tRNA的Northern印记杂交实验
发布: 2013年04月05日第3卷第7期 DOI: 10.21769/BioProtoc.464 浏览次数: 15389
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参见作者原研究论文
The authors used this protocol in:
Aug 2009
Abstract
tRNAs are small RNAs around 70-90 nt. tRNAs are different from many other small RNAs in that they are very abundant, which makes it difficult to study their transcriptional regulation by traditional northern blot. Traditional Northern blot involves incorporation of radioactive nucleotides through polymerization, however, tRNA is too short for polymerization. Traditional Northern blot detects changes in RNA levels, however, tRNA are so abundant that small changes in their levels will escape detection. For these reasons, metabolic labeling by radioactive Uracil has been used instead. However, metabolic labeling can only examine changes in total tRNA, but cannot distinguish different types of tRNAs. The following protocol describes a method to examine individual tRNA gene transcription by northern blot.
Keywords: TRNA (tRNA)
Northern Blot (Northern印迹)
Yeast (酵母)
Urea P (尿素P)
TRNA (tRNA)
Northern Blot (Northern印迹)
Yeast (酵母)
Urea PAGE (尿素页)
Small RNA (小核糖核酸)
Background
Materials and Reagents
- RapidGel (500 ml) (Affymetrix, catalog number: 75848 )
- Urea (CO(NH2)2) (Sigma-Aldrich, catalog number: U6504 )
- Tetramethylethylenediamine (TEMED, C6H16N2) (Thermo Fisher Scientific, catalog number: 110-18-9 )
- APS/ Ammonium persulfate ((NH4)2S2O8) (Sigma-Aldrich, catalog number: A3678 )
- Formamide (CH3NO) (Thermo Fisher Scientific, catalog number: 75-12-7 )
- DEPC/ Diethylpyrocarbonate (O(COOC2H5)2) (Sigma-Aldrich, catalog number: D5758 )
- Bromophenol Blue (C19H10Br4O5S) (Sigma-Aldrich, catalog number: B0126 )
- Xylene Cyanol FF (C25H27N2NaO6S2) (Sigma-Aldrich, catalog number: X4126 )
- Probe DNA oligo
- [γ-32P]-ATP, 10 Ci/mmol 2 mCi/ml, 250 μCi (PerkinElmer, catalog number: BLU002250UC )
- Whatman 3 M paper (Whatman, catalog number: 3 MM Chr )
- Microspin G-25 column (GE Healthcare, catalog number: 27-5325-01 )
- Hybond N+ membrane (Amersham, catalog number: RPN303B )
- Ficoll (Sigma-Aldrich, catalog number: F4375 )
- Sodium citrate
- BSA
- Polyvinlypyrrolidone (Sigma-Aldrich, catalog number: PVP360 )
- T4 Polynucleotide Kinase (New England Biolabs, catalog number: M0201S )
- 10x PNK buffer (New England Biolabs, catalog number: B0201S )
- Tris base (Thermo Fisher Scientific, catalog number: 77-86-1 )
- Boric acid (Thermo Fisher Scientific, catalog number: 10043-35-3 )
- EDTA (Sigma-Aldrich, catalog number: EDS-1KG )
- Methylene blue (Sigma-Aldrich, catalog number: 28514-100G )
- Sodium acetate (Sigma-Aldrich, catalog number: S2889 )
- Salmon sperm DNA (Life Technologies, InvitrogenTM, catalog number: 15632-011 )
Equipment
- Bench top centrifuge
- UV cross linker (Strata linker,model: 1800 )
- Power supply with constant voltage > 450 V
- Exposure cassette / intensifier screen (Sigma-Aldrich, catalog number: C5479-1EA )
- Hybridization bottle
- Hybridization oven
- Large gel cassette (around 20 x 40 cm)
- Film developer
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Wei, Y. (2013). Northern Blot of tRNA in Yeast. Bio-protocol 3(7): e464. DOI: 10.21769/BioProtoc.464.
Download Citation in RIS Format
分类
分子生物学 > RNA > RNA 检测
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