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Simple Purification of DNA from Plasmid Minipreps, PCR Amplifications,,,and Agarose Gels

High yields of extremely pure DNA with StrataPrep nucleic acid purification kits


Jeff Braman Scott Basehore
Stratagene Cloning Systems, Inc.


Stratagene announces a new line of DNA purification products, which provide reliable isolation of high-purity miniprep plasmid, PCR product and gel-fractionated DNAs. Purified miniprep DNA prepared with the StrataPrep plasmid miniprep kit is easily digested with restriction enzymes, is transformed with high efficiency and is accurately sequenced. With the StrataPrep PCR purification kit, PCR products are purified devoid of primers and small, nonspecific amplification products. These purified PCR products are cloned with high efficiency. Gel-isolated DNA that is purified using the StrataPrep DNA gel extraction kit is recovered with excellent yield and is efficiently cloned.

Purification of nucleic acids is a time-consuming part of many research projects. To eliminate this bottleneck, Stratagene has developed the StrataPrep nucleic acid purification kits. These products enable researchers to purify DNA from minipreps, PCR amplifications and agarose gels rapidly and with high yield and purity.

Nucleic acid purification using the StrataPrep products is based upon the principles discussed by Vogelstein and Gillespie.1 The kits utilize a special DNA-binding solid support that is manufactured in a microspin format. This format allows easy removal of contaminants by centrifugal washing, and the desired product is eluted in a small volume of buffer or water. The purified DNA is ready for a variety of applications.

Figure 1
Purification Protocols

StrataPrep Plasmid Miniprep Kit

  • Spin 1.5-ml overnight culture
  • Lyse cells
  • Spin
  • Transfer lysate to microspin cup, spin, wash and spin
  • Add elution buffer and spin to collect plasmid DNA

StrataPrep PCR Purification Kit

  • Add DNA binding reagent and transfer to microspin cup
  • Spin
  • Wash and spin
  • Add elution and spin to collect PCR Product

StrataPrep PCR Purification Kit

  • Add gel dissolution buffer to gel slice
  • Transfer to microspin cup
  • Spin
  • W ash and spin
  • Add elution buffer and spin to collect DNA

Figure 1 outlines the simple and rapid protocols used for purifying miniprep plasmid DNA, PCR products and agarose gel-fractionated DNA. Each microspin cup in the StrataPrep plasmid miniprep kit yields up to 20 g of purified plasmid DNA from overnight bacterial cultures of 1.5 to 3 ml. Each microspin cup in the StrataPrep PCR purification kit and DNA gel extraction kit yields as much as 10 g of purified DNA.

Plasmid Purification

figure 2

Plasmid DNA isolated with the StrataPrep plasmid miniprep kit exhibits OD260 -to-OD280 ratios between 1.8 and 2.0, indicating a high degree of nucleic acid purity.2 Starting with 30 individual 1.5- to 3-ml liquid cultures, purified plasmid DNA can be obtained in an average of 2.5 minutes per miniprep. Plasmid and cosmid DNAs isolated with the StrataPrep plasmid miniprep kit are easily digested with restriction enzymes (figure 2). Plasmid DNA purified with the StrataPrep kit transforms chemically competent E. coli with efficiencies equivalent to those obtained for DNA that has been purified using cesium chloride density gradients. For example, 100 pg of a 2.9-kb plasmid purified using either the StrataPrep kit or the cesium chloride density gradient method transformed Epicurian Coli XL2-Blue ultracompetent cells with an efficiency of 2 x 109 colony forming units (cfu)/g of plasmid DNA (data not shown).

figure 3

Sequence analysis of purified nucleic acids is a rigorous functional test of DNA purity. DNA purified with the StrataPrep kit was sequenced using fluorescent dye terminator and dye primer chemistries with an ABI PRISM 377 sequencer (figure 3). In this data set, only 3 sequence ambiguities were identified out of approximately 600 bases. In a separate sequencing experiment, plasmid DNA was sequenced using a LI-COR 4000L DNA sequencer and infrared-labeled sequencing primers. Sequence comparisons were made of plasmid DNA purified using either the StrataPrep kit or a silica-gel membrane method. Both sequences were aligned with a published data set. For the silica-gel membrane method, 12 ambiguous base calls were identified in 801 bases of sequence. Only 8 ambiguities were seen in 795 bases of sequence from plasmid that was purified using the StrataPrep plasmid miniprep kit (data available upon request).

Purification of PCR Products

Figure 4

The StrataPrep PCR purification kit is effective at removing primers and nonspecific amplification products smaller than 100 bp. Figure 4 shows that ethidium bromide-stained material smaller than 100 bp is eliminated from 0.6- and 1.5-kb PCR products using the StrataPrep kit (lanes 3 and 7, respectively), whereas the silica-gel membrane retained contaminants migrating between 72 and 118 bp (lanes 4 and 8, respectively).

figure 5

Cloning efficiency of the 1.5-kb PCR product purified using the StrataPrep ki t was compared to PCR product purified using the silica-gel membrane method (figure 5). A 1.5-kb PCR product was generated using amplification primers, each containing a different restriction site sequence. The PCR product was digested with both restriction enzymes, purified using each method, ligated to digested pBluescript SK(+) vector and transformed into XL2-Blue ultracompetent cells. In five separate experiments, the 1.5-kb PCR product purified using the StrataPrep PCR purification kit and cloned showed an average transformation efficiency of 9.7 x 106 cfu/g of vector DNA. The percent of clones that contained inserts averaged 90%. For the 1.5-kb PCR product purified and cloned using the silica-gel membrane method, the transformation efficiency was 5.5 x 106 cfu/g of vector DNA, and the percent of clones that contained inserts averaged 82%. Thus, purification using the StrataPrep kit resulted in nearly a 2-fold increase in cloning efficiency and a higher percent of clones with inserts.

Gel Isolation

Figure 6

Figure 6 shows the yield of DNA fragments that were recovered from a conventional 1% agarose gel using either the StrataPrep DNA gel extraction kit or the silica-gel-membrane method. The two methods appear to be equivalent for fragments ranging in size from 0.25 to 9 kb. A higher yield of the 23-kb fragment is obtained with the StrataPrep DNA gel extraction kit. Transformation efficiencies for ligated 1.5- and 0.6-kb PCR products that were isolated with the StrataPrep DNA gel extraction kit were 1.7 and 9.8 x 106 cfu/g, respectively. These values were equival ent to 1.5- and 0.6-kb PCR products purified using the silica-gel membrane method (data not shown). For PCR products that were purified using the StrataPrep DNA gel extraction kit, the percentage of clones with inserts (based on blue-white color selection) was 97% for the 1.5-kb fragment and 95% for the 0.6-kb fragment. The corresponding percent of clones with inserts for PCR products that were purified using the silica-gel membrane was 83% for the 1.5-kb PCR product and 92% for the 0.6-kb PCR product.

Conclusions

The StrataPrep plasmid miniprep, PCR purification and DNA gel extraction kits are reliable, simple and convenient to use. Excellent yield of high-purity DNA with these kits facilitates digestion, sequencing, cloning and other manipulations of DNA.

REFERENCES

  1. Vogelstein, B., and Gillespie, D. (1979) Proc. Nat. Acad. Sci. USA 76: 615-619.

  2. Sambrook, J., Fritsch, E.F., and Maniatis,T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.


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