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- 10× and 1× MOPS running buffer (see recipe for 10× buffer)
- 12.3 M (37%) formaldehyde, pH >4.0
- RNA sample: total cellular RNA ( appendix 1I ) or poly(A)+ RNA ( appendix 1I )
- Formamide
- Formaldehyde loading buffer (see recipe )
- 0.5 M ammonium acetate and 0.5 µg/ml ethidium bromide in 0.5 M ammonium acetate or 10 mM sodium phosphate (pH 7.0; see recipe )/1.1 M formaldehyde with and without 10 µg/ml acridine orange
- 0.24‐ to 9.5‐kb RNA ladder (Life Technologies) (optional)
- 0.05 M NaOH/1.5 M NaCl (optional)
- 0.5 M Tris⋅Cl (pH 7.4; appendix 2A )/1.5 M NaCl (optional)
- 20×, 2×, and 6× SSC ( appendix 2A )
- 0.03% (w/v) methylene blue in 0.3 M sodium acetate, pH 5.2 (optional)
- DNA suitable for use as probe (unit 5.1 ) or for in vitro transcription to make RNA probe (Table 5.17.1 )Table 5.7.1 MaterialsSelection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases Properties of Materials Used for Immobilization of Nucleic Acids b Properties of Materials Used for Immobilization of Nucleic Acids
Vector Size (bp) Markers a Promoters pBluescript 2950 amp, lacZ ′ T3, T7 pGEM series 2746‐3223 amp, lacZ ′ SP6, T7 pGEMEX‐1 4200 amp SP6, T3, T7 pSELECT‐1 3422 tet, lacZ ′ SP6, T7 pSP18, 19, 64, 65 2999‐3010 amp SP6 pSP70, 71, 72, 73 2417‐2464 amp SP6, T7 pSPORT1 4109 amp, lacZ ′ SP6, T7 pT3/T7 series 2700, 2950 amp, lacZ ′ T3, T7 pWE15 8800 amp, neo T3, T7 pWE16 8800 amp, dhfr T3, T7 Nitrocellulose Supported nitrocellulose Uncharged nylon Positively charged nylon Activated papers Application ssDNA, RNA, protein ssDNA, RNA, protein ssDNA, dsDNA, DNA, protein ssDNA, dsDNA, RNA, protein ssDNA, RNA Binding capacity (µg nucleic acid/cm2 ) 80‐100 80‐100 400‐600 400‐600 2‐40 Tensile strength Poor Good Good Good Good Mode of nucleic acid attachment c Noncovalent Noncovalent Covalent Covalent Covalent Lower size limit for efficient nucleic acid retention 500 nt 500 nt 50 nt or bp 50 nt or bp 5 nt Suitability for reprobing Poor (fragile) Poor (loss of signal) Good Good Good Commercial examples Schleicher & Schuell BA83, BA85;Amersham Hybond‐C Schleicher & Schuell Optibond;Amersham Hybond‐C extra Amersham Hybond‐N;Stratagene Duralon‐UV;Du Pont NEN GeneScreen Schleicher & Schuell Nytran;Amersham Hybond‐N+ ;Bio‐Rad ZetaProbe;PALL Biodyne B;Du Pont NEN GeneScreen Plus Schleicher & Schuell APT papers
a Abbreviations: amp, ampicillin resistance; dhfr, dihydrofolate reductase; lacZ , β‐galactosidase α‐peptide; neo, neomycin phosphotransferase (kanamycin resistance); tet, tetracycline resistance. - Formamide prehybridization/hybridization solution (see recipe )
- 2× SSC/0.1% (w/v) SDS
- 0.2× SSC/0.1% (w/v) SDS, room temperature and 42°C
- 0.1× SSC/0.1% (w/v) SDS, 68°C
- 55°, 60°, and 100°C water baths
- Oblong sponge slightly larger than the gel being blotted
- RNase‐free glass dishes (baked for 4 hr at 300°C)
- Whatman 3MM filter paper sheets
- UV‐transparent plastic wrap (e.g., Saran Wrap or other polyvinylidene wrap)
- Nitrocellulose or nylon membrane (see Table 5.17.2 for list of suppliers)Table 5.7.2 MaterialsSelection of Cloning Vectors Incorporating Promoters for Bacteriophage RNA Polymerases Properties of Materials Used for Immobilization of Nucleic Acids b Properties of Materials Used for Immobilization of Nucleic Acids
Vector Size (bp) Markers a Promoters pBluescript 2950 amp, lacZ ′ T3, T7 pGEM series 2746‐3223 amp, lacZ ′ SP6, T7 pGEMEX‐1 4200 amp SP6, T3, T7 pSELECT‐1 3422 tet, lacZ ′ SP6, T7 pSP18, 19, 64, 65 2999‐3010 amp SP6 pSP70, 71, 72, 73 2417‐2464 amp SP6, T7 pSPORT1 4109 amp, lacZ ′ SP6, T7 pT3/T7 series 2700, 2950 amp, lacZ ′ T3, T7 pWE15 8800 amp, neo T3, T7 pWE16 8800 amp, dhfr T3, T7 Nitrocellulose Supported nitrocellulose Uncharged nylon Positively charged nylon Activated papers Application ssDNA, RNA, protein ssDNA, RNA, protein ssDNA, dsDNA, DNA, protein ssDNA, dsDNA, RNA, protein ssDNA, RNA Binding capacity (µg nucleic acid/cm2 ) 80‐100 80‐100 400‐600 400‐600 2‐40 Tensile strength Poor Good Good Good Good Mode of nucleic acid attachment c Noncovalent Noncovalent Covalent Covalent Covalent Lower size limit for efficient nucleic acid retention 500 nt 500 nt 50 nt or bp 50 nt or bp 5 nt Suitability for reprobing Poor (fragile) Poor (loss of signal) Good Good Good 推荐方法- 肽聚糖 peptide glycan
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