Highly efficient purification of plasmid DNA by combining fast SDS/alkaline lysis of the starting material with the speed and purity of silica-membrane column purification.
pUC19 plasmid was isolated from 5 mL E. coli overnight LB cultures using the ISOLATE II Plasmid Mini Kit and cut with various restriction enzymes, before analysis on 1% TAE agarose gel. Each restriction digestion (lanes 1-6) was performed in triplicate on plasmids from different overnight cultures. The results illustrate consistency in plasmid recovery.
pUC19 plasmid was isolated from increasing volumes of E. coli from an overnight LB culture (0.05 ml, 0.1 mL, 1 mL, 3 mL and 5 mL culture, lanes 1- 5 respectively), using ISOLATE II Plasmid Mini Kit and run on 1.5% TAE agarose gel. The results illustrate the very high binding capacity of the columns in the ISOLATE II Plasmid Mini Kit.
pET19 plasmid was isolated from 3 mL E. coli overnight LB culture using ISOLATE II Plasmid Mini Kit. Sequencing was carried out based on standard T7 promoter and terminator primers, allowing more than 750 nucleotides to be read.
The ISOLATE II Plasmid Mini Kit provides a simple, efficient column-based method for the isolation of plasmid DNA from bacterial cultures, without the need for hazardous reagents such as phenol.
By combining SDS/alkaline lysis with the speed and ease-of-use of silica-membrane purification, the ISOLATE II Plasmid Mini Kit provides a fast method for the purification of high-quality plasmid DNA. Separate protocols are provided for the isolation of high-copy plasmids, the isolation of low-copy plasmid, P1 constructs and cosmid DNA from E. coli and the isolation of plasmids from gram-positive bacteria such as Bacillus, Staphylococcus, Bifidobacteria or Corynebacteria.
The ISOLATE II Plasmid Mini Kit has been designed to deliver optimal performance in cloning, sequencing as well as end-point PCR alongside any enzyme from the Meridian PCR portfolio, including MyTaq DNA Polymerase. Additionally, the ISOLATE II Plasmid Mini Kit can be used in tandem with the SensiFAST Real-Time PCR Kits for high-performance qPCR.
| Presentation | 10 Preps | 50 Preps | 250 Preps |
| ISOLATE II Plasmid Mini Spin Columns | 10 | 50 | 250 |
| Collection Tubes (2 mL) | 10 | 50 | 250 |
| Resuspension Buffer P1 | 5 mL | 15 mL | 75mL |
| Lysis Buffer P2 | 5 mL | 15 mL | 100 mL |
| Neutralization Buffer P3 | 5mL | 20 mL | 100 mL |
| Wash Buffer PW1 | 6 mL | 30 mL | 2 x 75 mL |
| Wash Buffer PW2 | 6 mL | 12 mL | 2 x 25 mL |
| Elution Buffer P | 13 mL | 13mL | 60 mL |
| RNase A (lyophilized) | 2.5 mg | 6 mg | 30 mg |
| Appearance | Colorless | ||
| Application | Cloning, Sequencing, Restriction digestion, Labeling, End-point PCR, qPCR, Transfection, Fluorescent sequencing, In vitro transcription | ||
| Sample type | E. coli and gram-positive bacteria such as Bacillus, Staphylococcus, Bifidobacteria or Corynebacteria. | ||
| Preparation time | Approximately 25 minutes for 18 preps | ||
| Plasmid size | < 15 kb | ||
| Elution volume | 50 µL | ||
| Yield | Up to 60 µg plasmid DNA | ||
| Storage | Store Resuspension Buffer P1 containing RNase A at 4°C and all other kit components at room temperature (18-25°C) | ||
| Functionality | Nucleic acid recovery >90% | ||
| RNase A | No detectable DNase activity | ||
Cat. No. Size
BIO-52055 10 Preps
BIO-52056 50 preps
BIO-52057 250 preps
A. Gupte, St. Vincent's Institute of Medical Research, Australia
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