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High Pure Plasmid Isolation Kit

Low to medium throughput, mini scale, plasmid isolation.

For life science research only. Not for use in diagnostic procedures.

Product No. Pack Size List Price Your Price Quantity
11754777001
50 purifications $ 90.00

Product No. Pack Size List Price Your Price Quantity
11754785001
250 purifications $ 364.00

The High Pure Plasmid Isolation Kit isolates purified plasmid DNA in small quantities using the alkaline lysis method, a commonly used method that generates highly purified plasmid DNA from E. coli, free of RNA contamination.

  • Quickly purify up to 24 plasmid samples in <30 minutes.
  • Minimize DNA loss with a kit that removes contaminants without precipitation or other handling steps that degrade DNA.
  • Improve reliability and reproducibility in downstream applications with a kit that removes RNA and other impurities that cause plasmid DNA to behave unpredictably.
  • Eliminate the use of hazardous organic compounds such as cesium chloride, phenol, chloroform, and ethidium bromide.

Contents

  1. Suspension Buffer
  2. RNase A, dry powder
  3. Lysis Buffer
  4. Binding Buffer
  5. Wash Buffer I
  6. Wash Buffer II
  7. Elution Buffer
  8. High Pure Spin Filter Tubes (containing glass fiber fleece)
  9. Collection Tubes

The High Pure Plasmid Isolation Kit prepares up to 15 μg purified plasmid DNA from bacterial cultures, that can be used directly in most molecular biology applications:

  • PCR
  • Sequencing (see Figure 1)
  • Cloning
  • In vitro transcription
  • Restriction enzyme digests
  • Random primed labeling

 

 

Figure 1:
Automated sequencing of plasmid DNA purified with the High Pure Plasmid Isolation Kit. A derivative of pUC 18, containing a 3.3 kb insert, was isolated from E. coli XL 1 blue (1.5 mL culture). Isolated plasmid DNA (250 ng) was sequenced using a fluorescently labeled sequencing primer. The sequence was analyzed using a LI-COR Automated DNA sequencer Model 4000 S, in auto sequencing mode.
Result: The sample was pure enough that more than 700 nucleotides from the insert could be read.

Nucleic acids bind to the surface of the glass fiber fleece in the presence of a chaotropic salt (guanidine HCl). This allows the High Pure filter tube to specifically immobilize nucleic acids (both DNA and RNA) while they are freed of contaminants.

Capacity: The High Pure Spin Filter Tubes hold up to 700 μL sample volume.
Sample: 0.5 - 4.0 mL cultures of E. coli (harvested at a density of 1.5 - 5.0 A 600 units/mL)  

Typical DNA Recovery



Starting Material Yield / Recovery Time Required Culture Volume Number of Reactions

E. coli XL1 blue,
pUC19

12 µg

30 minutes

2 mL

50 or 250

E. coli HB 101,
pUC19

6 µg

30 minutes

2 mL

 

E. coli DH5a,
pUC19

3.5 µg

30 minutes

2 mL

 


Principle

The kit relies on alkaline lysis to release plasmid DNA from bacteria. RNase removes all RNA in the lysate. After cellular debris and (entrapped) genomic DNA are removed by centrifugation, the remaining supernatant is mixed with a chaotropic salt and applied to the glass fiber fleece in a High Pure Spin Filter Tube. Under the buffer conditions used in the procedure, the plasmid binds to the glass fiber fleece, while contaminating substances (salts, proteins, and other cellular contaminants) do not. Brief wash-and-spin steps readily remove these contaminants. Once purified, the plasmid can be easily eluted in a small volume of low-salt buffer or water.


Quality

Plasmid pUC19 (4.5 µg) is purified from a 1.5 mL suspension of E. coli JM83, which was grown in LB-medium with ampicillin for 16 hours, to a cell density of 5 A 600 units/mL. 1 µg of the purified plasmid DNA is incubated for 1 hour at +37°C with 5 units of the restriction endonuclease Eco RI and then analyzed by agarose gel electrophoresis. The isolated plasmid DNA is as sensitive to restriction endonuclease digestion as plasmid DNA isolated by CsCl density centrifugation.

Product No: 11754777001
Product No: 11754785001

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Article (PDF, 773.44 KB)
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