Plasmid preparation

For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to plasmid preparation using the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of resultant plasmid solution is applied to agarose gel electrophoresis to examine the yields of plasmids. 

Three to several colonies for each sample are inoculated into the same 600 pL of LB medium containing 50 pg/mL of kanamycin and cultured at 37°C overnight. For glycerol stock, 50 pL of the culture is added to 25 pL of Cell Stock Buffer in a 96-well plate. The remaining suspensions are applied to the plasmid preparation with the Wizard SV 96 Plasmid DNA Purification System see Note 6). One microliter of the resultant plasmid solution is directly applied to agarose gel electrophoresis for estimation of the size of plasmid in a form of covalently closed circular. Similarly, another 1 pL of the plasmid solution is treated with S fi/Pmel and analyzed by agarose gel electrophoresis for an insert size check. This plasmid solution set is the final product and is reserved in a freezer. 

The plasmid preparation using the Wizard SV 96 Plasmid DNA Purification System follows the protocol from Promega, except that TE buffer instead of H O is used for the final plasmid elution buffer. This alteration is applied to all the plasmid preparation processes in this chapter. 

MAGNIA plasmid preparation robot MagExtractor MFX9600 (Toyobo). 

If you do not think that you have isolated the desired pUC19/4K plasmid, prepare a map of the plasmid that you think you have isolated. Support your drawing with an explanation of how the digestion of this plasmid with the various restriction enzymes would produce the results that you obtained. 

Redetermine the ODs of your plasmid preparations to ensure that equal amounts are used. 

Plasmid preparation to obtain linearized proviral HIV -1 DNA in which the entire RT or PRO is deleted. 

Plasmid DNA used contained the luciferase reporter gene (for in vitro experiments) including the CMV sequence. Plasmids present the normalized criteria of quality endotoxin level <20 EU/mg, supercoiled DNA >90%, E. coli-derived DNA contaminant <5%, RNA contaminant <5%, and protein contamination <1%. Such a plasmid preparation has been described (16). 

The target DNA can be plasmid preparations of cDNA clones, PCR-amplified inserts of cDNA clones, or PCR-amplified open reading frames (ORFs). The most suitable will depend on availability and ease of use. 

Dialysis of plasmid preparations is done with cellulose dialysis membranes with a mol wt cutoff of <12,000 (Spectropor, Spectrum Medical Industries, Houston, TX). 

The plasmid preparations used in these studies contained some level (<20%) of open circle forms prior to irradiation their number increased after low doses of radiation as they are produced from damaged supercoiled molecules. The increase in open circle forms ceases on exhaustion of the supply of parent supercoiled molecules. Further radiation exposure resulted in damage to the open circle molecules revealed as an exponential decrease in open circle forms (Fig. 2). Analysis of the amount of open circle forms is described by the difference of two exponentials - one due to the production and the other due to the destruction of the open circle form. The complexity of these curves presents difficulties in resolving the two exponential components (9) since only some of the data points are used in each fit. Combining data from several experiments increases the number of data points, enabling more accurate target determinations. 

DNA template preparations are a major source of contamination in cell-free synthesis. In vitro translation reactions are sensitive to contamination by salts, RNases, detergents, and alcohol, all of which are typically used in commercial plasmid purification kits. As such, plasmids prepared by resin-based purification protocols must be further purified by phe-nol/chloroform and chloroform extraction. DNA should be precipitated with isopropanol, washed in ethanol, dried, and taken up in RNase-free water to a concentration of about 1 mg mL . The DNA should be stored frozen, in small aliquots. 

The I-Scel vector to be injected should be designed such that the expression cassette of interest (e.g., including promoter, transgene, and polyadenylation signal) is flanked by two I-Scel recognition sites (Fig. 1). The expression cassette of the SB vector is flanked by two SB IRs and DRs (Fig. 2). The plasmid DNA should be prepared and purified using a high-purity plasmid preparation kit. DNA concentration and purity can be checked by spectrometry. The ratio of A,7A,. should be between 1.8-2.0. 

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