» Sign in / Register

Bacteria DNA Preparation Kit

Spin column based genomic DNA purification from bacteria

Product Cat. No. Amount Price (EUR) Buy / Note
Bacteria DNA Preparation Kit PP-214XS 10 preparations 20,00 Add to Basket/Quote Add to Notepad
Bacteria DNA Preparation Kit PP-214S 50 preparations 80,00 Add to Basket/Quote Add to Notepad
Bacteria DNA Preparation Kit PP-214L 250 preparations 320,00 Add to Basket/Quote Add to Notepad

For in vitro use only!

Shipping: shipped at ambient temperature

Storage Conditions: store at ambient temperature

Shelf Life: 12 months

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.

Kit contents:
Resuspension Buffer
Lysis Buffer
Binding Buffer
RNase A (before use, add double distilled water as indicated on the bottle) - store at -20 °C
Lysozyme (before use, add double distilled water as indicated on the bottle) - store at -20 °C
Proteinase K (before use, add double distilled water as indicated on the tube) - store at -20 °C
Activation Buffer
Washing Buffer (before use, add 96-99 % Ethanol as indicated on the tube)
Elution Buffer
Spin Columns and 2 ml Collection Tubes

To be provided by you:
96-99 % Ethanol
Double distilled water
Microtubes 1.5 or 2.0 ml

Applications:
The obtained DNA is suitable for a variety of applications, including real-time PCR, southern blot analysis, genotyping and discovery or validation of SNP/SSR markers.
Before start, add the following components (not included in the kit) as indicated on the respective bottle/tube:

  • double distilled water to RNase A, Lysozyme and Proteinase K
  • 96-99 % Ethanol to the Washing Buffer

Preparation procedure:
For XS pack (10 preps): Before start, add 100 μl dd-water to the Proteinase K tube, 25 μl dd-water to the Lysozyme tube, 30 μl dd-water to the RNase A tube and 9.6 ml 96-99 % Ethanol (not included in the kit) to the Washing Buffer bottle.
For S pack (50 preps): Before start, add 500 μl dd-water to the Proteinase K tube, 125 μl dd-water to the Lysozyme tube, 150 μl dd-water to the RNase A tube and 48 ml 96-99 % Ethanol (not included in the kit) to the Washing Buffer bottle.
For L pack (250 preps): Before start, add 500 μl dd-water to each Proteinase K tube, 125 μl dd-water to each Lysozyme tube, 150 μl dd-water to each RNase A tube and 120 ml 96-99 % Ethanol (not included in the kit) to each Washing Buffer bottle.

BufferPP-214XS
10 preps
PP-214S
50 preps
PP-214L
250 preps
Resuspension Buffer3.2 ml16 ml80 ml
Lysis Buffer3.2 ml16 ml80 ml
Binding Buffer3.2 ml16 ml80 ml
RNase A
(50 mg/ml)
1.5 mg7.5 mg5x 7.5 mg
Lysozyme
(100 mg/ml)
2.5 mg12.5 mg5x 12.5 mg
Poteinase K
(10 mg/ml)
1 mg5 mg5x 5 mg
Activation Buffer1.2 ml6 ml30 ml
Washing Bufferadd 9.6 ml Ethanol
(final volume 12 ml)
add 48 ml Ethanol
(final volume 60 ml)
add 120 ml Ethanol to each bottle (final volume 150 ml each)
Elution Buffer1 ml5 ml25 ml

It is essential to use the correct amount of starting material in order to obtain optimal DNA yield and purify. A maximum amount of 108 bacteria cells can generally be processed. Overnight cultured bacteria cells can be processed. Cell pellets can be stored at -70 °C for several months.

1. Preparation from Gram-positive bacteria
A Cell Resuspension:

  • Harvest 500 μl of cultured bacteria cells by centrifugation at 10,000 g for 1 min
  • Discard the supernatant
  • Resuspend the cell pellet in 300 μl of Resuspension Buffer
  • Add 2 μl of Lysozyme Solution
  • Mix well by inverting several times
  • Incubate tube at 37 °C for 1 hour
  • Centrifuge at 10,000 g for 1 min
  • Discard the supernatant


B Cell Lysis:

  • Add 300 μl Lysis Buffer and 2 μl RNase A to cell pellet
  • Vortex vigorously for 30-60 sec
  • Add 8 μl Proteinase K and mix by pipetting
  • Incubate at 60 °C for 10 min and cool down for 5 min
  • Add 300 μl Binding Buffer and vortex briefly
  • Place the tube on ice for 5 min
  • Centrifuge for 5 min at 10,000 g


C Column Activation [optional]:

  • Place a spin column into a 2 ml collection tube
  • Add 100 μl Activation Buffer into the Spin Column
  • Centrifuge at 10,000 g for 30 sec and immediately proceed to next step
  • Discard the flow-through


D Column Loading:

  • Pipet the supernatant directly into the spin column
  • Centrifuge for 1 min at 10,000 g
  • Discard the flow-through


E Primary Washing:

  • Add 500 μl Washing Buffer into spin column
  • Centrifuge for 30 sec at 10,000 g
  • Discard the flow-through


F Secondary Washing:

  • Add 500 μl Washing Buffer into the spin column.
  • Centrifuge for 30 sec at 10,000 g
  • Discard the flow-through
  • Centrifuge again at 10,000 g for 1 min to remove residual Washing Buffer
  • Discard the 2 ml wash tube and place the column in the elution tube


G Elution of DNA:

  • Add 40-50 μl Elution Buffer into the center of the column
  • Incubate at room temperature for 1 min
  • Centrifuge at 10,000 g for 2 min
  • Store DNA at 4 °C or -20 °C


2. Preparation from Gram-negative bacteria
A Cell Lysis:

  • Harvest 500 μl of cultured bacteria cells by centrifugation at 10,000 g for 1 min
  • Discard the supernatant
  • Add 300 μl Lysis Buffer and 2 μl RNase A to cell pellet
  • Vortex vigorously for 30-60 sec
  • Add 8 μl Proteinase K and mix by pipetting
  • Incubate at 60 °C for 10 min and cool down for 5 min
  • Add 300 μl Binding Buffer and vortex briefly
  • Place the tube on ice for 5 min
  • Centrifuge for 5 min at 10,000 g


B Column Activation [optional]:

  • Place a spin column into a 2 ml collection tube
  • Add 100 μl Activation Buffer into the Spin Column
  • Centrifuge at 10,000 g for 30 sec and immediately proceed to next step
  • Discard the flow-through


C Column Loading:

  • Place a spin column into a 2 ml collection tube
  • Pipet the lysate directly into the spin column
  • Centrifuge for 1 min at 10,000 g
  • Discard the flow-through


D Primary Washing:

  • Add 500 μl Washing Buffer into spin column
  • Centrifuge for 30 sec at 10,000 g
  • Discard the flow-through


E Secondary Washing:

  • Add 500 μl Washing Buffer into the spin column
  • Centrifuge for 30 sec at 10,000 g
  • Discard the flow-through
  • Centrifuge again at 10,000 g for 1 min to remove residual Washing Buffer
  • Discard the 2 ml wash tube and place the column in the elution tube


F Elution of DNA:

  • Add 40-50 μl Elution Buffer into the center of the column
  • Incubate at room temperature for 1 min
  • Centrifuge at 10,000 g for 2 min
  • Store DNA at 4 °C or -20 °C

Availability Restriction: Exclusively distributed in Japan by Greiner BioOne