Linear plasmid DNA

FIGURE 13.2 Foreign DNA sequences can be inserted into plasmid vectors by opening the circnlar plasmid with a restriction endonnclease. The ends of the linearized plasmid DNA are then joined with the ends of a foreign sequence, reclosing the circle to create a chimeric plasmid. 

Fig. 14. lEC of supercoiled, relaxed, and linear plasmid DNA. Column Nucleogen DEAE-4000 (6 X125 mm) sample 4 of supercoiled plasmid pBR 322, 1.5 /tg of the plasmid pBR 322 linearized with the restriction endonuclease Bam HI the relaxed form is generated from the supercoiled form during storage chromatographic conditions linear gradient from 750 to 1500 mM KO in 120 min 20 mM K-phosphate, 5 M urea, pH 6.6 1.5 ml/min 45 bar ambient temperature. From [19]. 

Synthesize the RNA. Detailed protocols for in vitro transcription have been published (Melton et ai, 1984 Krieg and Melton, 1987 Terns et ai, 1992). Typically, 0.2-1 pg of linearized plasmid DNA or 50-100 ng of PCR fragment (the exact amount depends on size of DNA fragment) are employed in a reaction volume of 10-20 /il for maximal transcription. Once the incubation is complete, the DNA template is removed from the reactions by digesting with RNase-free DNase. Proteins in the reactions are removed by extraction with an equal volume of phenol chloroform isoamyl alcohol (25 24 1), and the unincorporated nucleotides present in the aqueous phase are removed... 

In contrast to the above well-known strategies to modify CNTs for DNA delivery, Geyik et al. recently developed a new approach using the covalent attachment of linearized plasmid DNA to MWNTs. Cifuentes-Rius et al. demonstrated that two different monomers, pentafluorophenyl methacrylate (PFM) and allylamine (AA), could be polymerized on the CNT surface in a home-built plasma reactor, allowing the formation of CNT-mediated gene delivery vectors. ... 

Long RNA probes are generated by in vitro transcription from linearized plasmid DNA containing a promoter sequence for a DNA-dependent RNA polymerase such as SP3, T3, or T7 polymerases. Commercially available kits for high-yield transcription with label incorporation are available. It is also technically trivial to introduce a T7 promoter sequence as a 5 -extension of a PCR primer. Amplification introduces the promoter sequence into the amplicon. Transcription of the amplicon with T7 RNA polymerase and a modified NTP yields the labeled RNA probe. Shorter RNA probes (less than 50 bases) are chemically synthesized, and label is most conveniently introduced during synthesis. 

Separation of supercoiled, open circular and linear plasmid DNA 204... 

Figure 12. Analytical anion-exchange HPLC of supercoiled and linearized plasmid DNA on a DEAE column. Sample, 0.19 supercoiled pUC8 DNA and 0.33 /tg pUC8 DNA linearized with icoRI column, Nucleogen DEAE-4000 (6 x 125 mm) apparatus, Merck-Hitachi 655 A-12 with a Meick-Hitachi 6S5A-22 spectrophotometer set at 260 nm with 0.08 AUFS elution, linear gradient from 0.66 to 1.2 M NaCI in 0.03 M sodium phosphate. pH 6.0, 5 M urea in 60 min flow-rate, 2 ml/min. Supercoiled plasmid DNA is eluted before (at 11.5 min) linear plasmid DNA (at 13.5 min). The minute peak following peak for supercoiled plasmid DNA represents relaxed plasmid DNA.

It seems not to unrealistic to predict that with the proper choice of chromatographic conditions (concentrations of denaturing agent, temperature, pH, etc.) it will become a routine matter to achieve separation of the complementary strands of open circular or linear plasmid DNA by HPLC. Of particular interest in this respect is the MonoQ column packing material, which can tolerate alkaline pH. 

Enzyme amount and incubation time are critical parameters for successful digestions. For example, too much enzyme will only contribute, on gel analysis, to the smearing of DNA after only 5 to 10 min incubation. For best results, a given batch of Bal31 enzyme should be calibrated for its digestion rate with a standard substrate such as linearized plasmid DNA or, preferably, with the target DNA. 

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