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    Cat. No.
    Size
    List Price*
    QTY
    BIO-52055
    10 preps
    S$28.00
    +
    BIO-52056
    50 preps
    S$88.00
    +
    BIO-52057
    250 Preps
    S$370.00
    +
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    Description

    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.

    Product Highlights

    • Fast - streamlined protocol for extraction of eighteen plasmid samples in as little as 15 minutes
    • Efficient – rapid SDS/alkaline lysis and efficient column-binding for high-yield recovery of all plasmid molecules
    • High-performance – efficient extraction of high-purity plasmid DNA, ideal for use in all downstream applications
    • Flexible – reliable recovery of high- and low-copy plasmids from all gram-positive bacteria

    Product Description

    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 Bioline 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.

    Applications

    • Cloning
    • Sequencing
    • Restriction digestion
    • Labeling
    • End-point PCR
    • qPCR
    • Transfection
    • Fluorescent sequencing
    • In vitro transcription
    Main
    Fast purification of high-quality plasmid DNA from bacteria.

    Nucleic Acid Isolation Guide

    Download the ISOLATE II Guide with detailed product descriptions and performance data to help you choose the best product for your research

    Specification

    Components

    Reagent

    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

    75 mL

    Lysis Buffer P2

    5 mL

    15 mL

    3 x 25 mL

    Neutralization Buffer P3

    5 mL

    20 mL

    100 mL

    Wash Buffer PW1

    6 mL

    30 mL

    2 x 75 mL

    Wash Buffer PW2

    2 mL

    2 x 6 mL

    2 x 20 mL

    Elution Buffer P

    5 mL

    15 mL

    75 mL

    RNase A (lyophilized)

    2 mg

    6 mg 

    30 mg

    Product Manual

    1

    1

    1

    Bench Protocol Sheet

    1

    1

    1

    Storage & Stability

    All kit components should be stored dry and at room temperature. When stored under the recommended conditions and handled correctly, full activity of kit reagents is retained until the expiry date indicated on the outer box label.

    Note: RNase A is supplied lyophilized. On addition of Resuspension Buffer P1, the RNase A solution should be kept stored at 4°C. When stored at 4°C. the RNase A solution is stable for at least 6 months.

    Shipping conditions

    Ambient temperature.



    FAQs

    The columns supplied with the ISOLATE II kits may appear similar, but each type has been optimized to work within the buffer system supplied with the corresponding kit. The swapping of columns (or buffers) between kits may lead to no recovery of nucleic acid whatsoever, or at the very least severely impaired purification.

    Interruption of the DNA/RNA extraction process is possible after the sample lysis only. It is possible to homogenize and lyse the samples and to store them in the freezer until use for RNA extraction. RNA clean up with a column cannot be interrupted and we recommend to avoid delays during the column purification process. If a delay is unavoidable the columns should be stored on ice.

    The elution buffer is ideal for applications such as restriction enzyme digestion, sequencing and PCR. It is possible to use DNase-free water for elution, but you should expect a slightly lower yield.

    The elution buffer of this kit does not contain EDTA which could interfere with downstream applications like PCR or sequencing.

    The high quality of the columns used in the ISOLATE II kits, including the ISOLATE II Plasmid Mini Kit, allow larger volumes of culture media to be used as starting material. The exact volume to be used depends on the strain and plasmid used in the experiment, but the ISOLATE II Plasmid Mini Kit columns will cope with the biomass from 5 mL of E. coli grown in LB to an OD600 of around 3. When using larger volumes of culture medium, it is important to ensure that as much spent medium as possible is removed from above the biomass pellet after spinning down the culture. If care is taken, up to 15 mL of bacterial culture medium can be used as a starting material, which should give a better yield of low-copy plasmid DNA. However, double the volumes of buffers P1, P2 and P3 should be used compared to the standard protocol.

    The most frequent cause of low yield is that the biomass pellet is not resuspended properly prior to lysis. Clumps of bacteria mean that the lysis solution cannot access all the cells and these unlysed cells are then spun down later in the process. Low yield can also be caused by incomplete transfer of the cleared lysate to column, especially when the white precipitate made on the addition of buffer P3 is loose, making it hard to retrieve the maximum amount of lysate. Very high biomass concentrations can also lead to low plasmid yield. This can be because the column becomes overloaded, so the yield per mg biomass is less than expected. However use of rich media such as SOC or TY instead of LB can give high biomass but very low plasmid concentration.

    The ISOLATE II Plasmid Mini Kit can be used to both purify and concentrate naked plasmid DNA. The DNA can be loaded directly onto a fresh column then the remaining washing, drying and elution steps followed as for the standard protocol. The sample can be concentrated by reducing the elution volume in the final stage. However, a better recovery can be obtained by using the ISOLATE II PCR and Gel Kit and treating the plasmid sample as though it were a PCR product.

    For the optimal recovery of large RNA/DNA fragments it is necessary to optimize the elution step. To increase the recovery rate it would be helpful to use a larger volume for elution, incubate the column with the elution buffer prior to centrifugation and use repeated elution steps. To avoid excessive dilution it may be helpful to reapply the eluted fraction again. Furthermore it could be helpful for DNA elution to heat the elution buffer to 70°C.