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GENOMIC DNA PREP KIT for BACTERIA

ORDERING INFORMATION

Catalog #

Product

Amount

Price (EUR)

# GEN 50 Bac
Genomic DNA Prep Kit for Bacteria
50 preparations


# GEN 250 Bac
Genomic DNA Prep Kit for Bacteria

250 preparations


PRODUCT COMPONENTS

Buffer

# GEN 50 Bac

# GEN 250 Bac

Bacteria Lysis Buffer

20 ml

100 ml

Binding Buffer

25 ml

125 ml

Gram(+)-Bacteria Buffer

20 ml

100 ml

Proteinase K (final conc. 10 mg/ml)

5 mg

5 x 5 mg

Lysozym (final conc. 100 mg/ml)

12.5 mg

5 x 12.5 mg

Column Activation Solution

6 ml

30 ml

Elution Buffer

5 ml

25 ml

Spin Columns & 2ml Tubes

50

250

DESCRIPTION

The spin column based Bacteria DNA Preparation Kit is designed for rapid and high purity isolation of genomic DNA from Gram-positive and Gram-negative bacteria. The spin column based method completely removes PCR inhibitors such as divalent cations and proteins resulting in a high purity preparation of genomic DNA. There is no use of phenol or chloroform, handling is safe and does not produce any harmful waste. Column based genomic DNA purification kits yield up to 30 μg DNA sized from 200 bp to 50 kb per preparation.

FEATURES
  • Spin column type
  • Purification size: 200 bp - 50 kb
  • DNA amounts up to 30 g

APPLICATIONS

  • real-time PCR
  • PCR
  • southern blot analysis
  • genotyping
  • discovery or validation of SNP/SSR marker

BASIC PROTOCOL

Before start, prepare the following components:

  • Add double distilled water to LysozymLysozym solution should be stored at -20C.
    • for 50 preparations (# GEN 50 Bac): add 125 l dd-water to 12.5 mg Lysozym.
    • for 250 preparations (# GEN 250 Bac): add 125 l dd-water to each tube of 12.5 mg Lysozym.
  • Add double distilled water to Proteinase K. Proteinase K solution should be stored at -20C.
    • for 50 preparations (# GEN 50 Bac): add 500 l dd-water to 5 mg Proteinase K.
    • for 250 preparations (# GEN 250 Bac): add 500 l dd-water to each tube of 5 mg Proteinase K.
  • 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 (# GEN 50 Bac): 60 ml
    • for 250 preparations (# GEN 250 Bac): 300 ml

A. DNA PREPARATION FROM GRAM-POSITIVE BACTERIA

1. Cell Resuspension

  • Harvest 500 l of cultured bacteria cells by centrifugation at 12,000 g for 1 min.
  • Proceed immediately.
  • Resuspend the cell pellet in 300 μl of Gram(+)-Bacteria Buffer.
  • Add 2 μl of Lysozym solution.
  • Mix well by inverting several times.
  • Incubate tube at 37C for 1 hour.
  • Centrifuge at 12,000 g for 1 min.
  • Discard the supernatant.

2. Cell Lysis

  • Add 350 l Bacteria Lysis Buffer to cell pellet.
  • Vortex vigorously for 30-60 sec.
  • Add 8 μl Proteinase K solution to cell lysate.
  • Mix by pipetting.
  • Incubate at 70C for 10 min and cool down for 5 min.
  • Centrifuge for 5 min at 12,000 g at room temperature.
  • Transfer supernatant to a new 1.5 ml microcentrifuge tube.

3. Adding Binding Buffer

  • Add 400 l Binding Buffer to the cell lysate.
  • Vortex vigorously for 30-60 sec.
  • Centrifuge for 5 min at 12,000 g at room temperature.

4. 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 10,000 g for 30 sec in a microcentrifuge.

5. Column Loading

  • Pipette the lysate directly into the Spin Column.
  • Centrifuge for 1 min at 12,000 g.
  • Discard the flow-through.

6. Primary Washing

  • Add 500 l Washing Buffer (80% Ethanol) into the Spin Column.
  • Centrifuge for 30 sec at 12,000 g.
  • Discard the flow-through.
7. Secondary Washing
  • Add 500 l Washing Buffer into the Spin Column.
  • Centrifuge for 30 sec at 12,000 g.
  • Discard the flow-through.
  • Centrifuge again at 12,000 g for 1 min to remove residual Washing Buffer.
  • Discard the 2 ml wash tube and place the column in the elution tube.

8. Elution of DNA

  • Add 40-50 μl Elution Buffer to the center of the column.
  • Incubate at room temperature for 1 min.
  • Centrifuge at 12,000 g for 2 min to elute DNA.
  • Store DNA at 4C (for prolonged storage, keep it at -20C or -80C).

B. DNA PREPARATION FROM GRAM-NEGATIVE BACTERIA

1. Cell Lysis

  • Harvest 500 l of cultured bacteria cells by centrifugation at 12,000 g for 1 min.
  • Proceed immediately.
  • Add 350 l Bacteria Lysis Buffer to cell pellet.
  • Vortex vigorously for 30-60 sec.
  • Add 8 l Proteinase K solution to cell lysate.
  • Mix by pipetting.
  • Incubate at 70C for 10 min and cool down for 5 min.
  • Centrifuge for 5 min at 12,000 g at room temperature.
  • Transfer supernatant to a new 1.5 ml microcentrifuge tube.

2. Adding Binding Buffer

  • Add 400 l Binding Buffer to the cell lysate.
  • Vortex vigorously for 30-60 sec.
  • Centrifuge for 5 min at 12,000 g.

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 10,000 g for 30 sec in a microcentrifuge.

4. Column Loading

  • Pipette the lysate directly into the Spin Column.
  • Centrifuge for 1 min at 12,000 g.
  • Discard the flow-through.

5. Primary Washing

  • Add 500 l Washing Buffer (80% Ethanol) into the Spin Column.
  • Centrifuge for 30 sec at 12,000 g.
  • Discard the flow-through.
6. Secondary Washing
  • Add 500 l Washing Buffer into the Spin Column.
  • Centrifuge for 30 sec at 12,000 g.
  • Discard the flow-through.
  • Centrifuge again at 12,000 g for 1 min to remove residual Washing Buffer.
  • Discard the 2 ml wash tube and place the column in the elution tube.

7. Elution of DNA

  • Add 40-50 μl Elution Buffer to the center of the column.
  • Incubate at room temperature for 1 min.
  • Centrifuge at 12,000 g for 2 min to elute DNA.
  • Store DNA at 4C (for prolonged storage, keep it at -20C or -80C).

STORAGE

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

Proteinase K solution and Lysozym solution should be stored at -20C.

For long term storage place Proteinase K lyophilisate and Lysozym lyophilisate at -20C.