15 Cloning Vectors
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16. Resuspend the pellet in 36 J.ll of distilled water.
17. Add 4 J.ll ofRNase A (1 mg/ml) and incubate 30 min at 37°C. If you wish
to digest the DNA with restriction enzymes, the restriction buffer and
restriction enzyme can be added together with the RNase A and the
reaction carried out at the same time.
Subprotocol 5
Chromatographie Method for Plasmid Preparation
Another plasmid isolation method makes use of the low molecular weight
and different binding properties of plasmid DNA compared to chromosomal DNA (Birnboim 1979, Birnboim 1983). The DNA solution is passed
through columns packed with different absorbents (e.g. Sepharose, Dextrane) that reversibly bind DNA. In a second step the DNA is eluted in fractions thereby separating plasmid DNA from other forms ofDNA, RNA and
protein. In the past several years kits have become commercially available
making use of the above-described technique. The absorbent material has
been further developed enabling large-scale plasmid preparations in high
purities. One of those kits used by the authors will be described in the following.
Materials
• A single colony of an E. coli strain (e.g. XL1-Blue, DH5a)
• QIAGEN-tip 500 (by QIAGEN Inc.)
• RNase A (10 mg/ml in 5 mM Tris-HCl (pH 8.0), heated to 70°C for 10 min
or lyophilized RNase A from the Qiagen kit)
• LB medium (see Subprotocol 1, Materials)
• Buffer P1 (50 mM Tris/HCl, 10 mM EDTA (pH 8.0))
• Buffer P2 (0.2 M NaOH, 1 o/o SDS)
• Buffer P3 (3.0 M potassium-acetate (pH 5.5))
• Buffer QBT (750 mM NaCl, 50 mM MOPS, 15o/o ethanol (pH 7.0), 0.15%
Triton X-100)
• Buffer QC (1.0 M NaCl, 50 mM MOPS, 15o/o ethanol (pH 7.0))
Reagents
Salutions
247
16. Resuspend the pellet in 36 J.ll of distilled water.
17. Add 4 J.ll ofRNase A (1 mg/ml) and incubate 30 min at 37°C. If you wish
to digest the DNA with restriction enzymes, the restriction buffer and
restriction enzyme can be added together with the RNase A and the
reaction carried out at the same time.
Subprotocol 5
Chromatographie Method for Plasmid Preparation
Another plasmid isolation method makes use of the low molecular weight
and different binding properties of plasmid DNA compared to chromosomal DNA (Birnboim 1979, Birnboim 1983). The DNA solution is passed
through columns packed with different absorbents (e.g. Sepharose, Dextrane) that reversibly bind DNA. In a second step the DNA is eluted in fractions thereby separating plasmid DNA from other forms ofDNA, RNA and
protein. In the past several years kits have become commercially available
making use of the above-described technique. The absorbent material has
been further developed enabling large-scale plasmid preparations in high
purities. One of those kits used by the authors will be described in the following.
Materials
• A single colony of an E. coli strain (e.g. XL1-Blue, DH5a)
• QIAGEN-tip 500 (by QIAGEN Inc.)
• RNase A (10 mg/ml in 5 mM Tris-HCl (pH 8.0), heated to 70°C for 10 min
or lyophilized RNase A from the Qiagen kit)
• LB medium (see Subprotocol 1, Materials)
• Buffer P1 (50 mM Tris/HCl, 10 mM EDTA (pH 8.0))
• Buffer P2 (0.2 M NaOH, 1 o/o SDS)
• Buffer P3 (3.0 M potassium-acetate (pH 5.5))
• Buffer QBT (750 mM NaCl, 50 mM MOPS, 15o/o ethanol (pH 7.0), 0.15%
Triton X-100)
• Buffer QC (1.0 M NaCl, 50 mM MOPS, 15o/o ethanol (pH 7.0))
Reagents
Salutions