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PLASMID MINI-PREP KIT - Column Type

ORDERING INFORMATION

Catalog #

Product

Amount

Price (EUR)

# PLA 50
ROVALAB Plasmid Mini-Prep Kit
50 preparations

43,50

# PLA 250
ROVALAB Plasmid Mini-Prep Kit

250 preparations

176,50

PRODUCT COMPONENTS

Buffer

# PLA 50

# PLA 250

Resuspension Buffer

16 ml

80 ml

RNase A

2 mg

5 x 2 mg

Lysis Buffer

16 ml

80 ml

Neutralization Buffer

20 ml

100 ml

Column Activation Solution

6 ml

30 ml

Elution Buffer

6 ml

30 ml

Spin Columns & 2ml Tubes

50

250

DESCRIPTION

ROVALAB Plasmid Mini-Prep Kit is designed for isolation of high-purity plasmid or cosmid DNA from cells for subsequent sequencing, restriction digests, or transformations. The 2-step alkaline lysis procedure and spin column based preparation provide a fast, easy and efficient way of DNA isolation without shearing or significant loss of product. It allows elution in a small volume of low-salt buffer. Time-consuming phenolchloroform extraction or alcohol precipitation is eliminated. The kit can be used either in micro-centrifuges or on vacuum manifolds and allows the extraction of up to 20 g DNA per preparation.

FEATURES

  • Spin column type
  • Binding capacity: 20 g DNA/column
  • Quick mini prep of pure plasmid
  • Sequencing & transfection grade
  • Low copy plasmid, large size circular DNA (formid, BAC clones) prep

APPLICATIONS

  • General sequencing
  • Transfection
  • Cloning

BASIC PROTOCOL

The DNA purification follows a simple binding, washing and eluting procedure. The optional secondary washing step minimizes the salt content of the purification product.

Before start, prepare the following components:

  • Add total RNase A to the Resuspension Buffer and mix well. RNase A containing Resuspension Buffer should be stored at 4C.
    • for 50 preparations (# PLA 50): add 2 mg RNase A to 16 ml Resuspension Buffer
    • for 250 preparations (# PLA 250): add 5 x 2 mg RNase A to 80 ml Resuspension Buffer
  • Prepare an 80% Ethanol (V/V) solution as Washing buffer. Please note that the Ethanol concentration of Washing Buffer may decrease during long time storage resulting in a possible drop of the final DNA yield.
    • for 50 preparations (# PLA 50): 80 ml
    • for 250 preparations (# PLA 250): 400 ml

1. Cell Harvest and Suspension

  • Harvest the bacterial cell culture (1-3 ml) by centrifugation.
  • Resuspend pelleted bacterial cells in 300 μl Resuspension Buffer containing RNase A by pipetting. 

2. Cell Lysis and Neutralization

  • Add 250 l Lysis Buffer and mix gently by inverting the tube 4-6 times (do not vortex!).
  • Add 350 l of Neutralization Buffer and invert immediately 4-6 times.
  • Centrifuge at 12,000 g for 10 min at room temperature in a microcentrifuge.

3. Column Activation

  • Place a Spin Column into a 2 ml collection tube.
  • Add 100 l of Column Activation Solution into the Spin Column.
  • Centrifuge at 12,000 g for 30 sec in a microcentrifuge.

4. Column Loading

  • Apply the supernatant from step 2 into the activated Spin Column by decanting or pipetting.
  • Centrifuge at 12,000 g for 30 sec in a microcentrifuge.
  • Discard the flow-through.

5. Column Washing

  • Place the DNA loaded Spin Column into the used 2 ml tube.
  • Apply 700 l of Washing Buffer (80% Ethanol) to the Spin Column.
  • Centrifuge at 12,000 g for 30 sec and discard the flow-through.
  • Optional Secondary Washing: Recommended only for DNA >200 bp, if highly purified DNA (for DNA sequencing, transfection etc.) is required.
    • Add 700 l of Washing Buffer to the Spin Column.
    • Centrifuge at 12,000 g for 30 sec and discard the flow-through.
  • Centrifuge again for 2 min to remove residual Washing Buffer.

6. Elution

  • Place the Spin Column into a clean 1.5 ml microtube (not provided in the kit).
  • Add 30-50 μl Elution Buffer to the center of the column membrane.
  • Incubate at room temperature for 1 min.
  • Centrifuge at 12,000 g for 1 min to elute DNA.

STORAGE

Store at room temperature (15 25 C) for 12 months.

For long term storage place RNase A lyophilisate at -20C.

RNase A containing Resuspension Buffer should be stored at 4C.