ORDERING INFORMATION
|
Catalog # |
Product |
Amount |
Price (EUR) |
| # GEN 50 Bac |
Genomic DNA Prep Kit for Bacteria |
50 preparations |
|
| # GEN 250 Bac |
|
250 preparations |
PRODUCT COMPONENTS
|
# 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 µg
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 Lysozym. Lysozym solution should be stored at -20°C.
-
for 50 preparations (# GEN 50 Bac): add 125 µl dd-water to 12.5 mg Lysozym.
-
for 250 preparations (# GEN 250 Bac): add 125 µl dd-water to each tube of 12.5 mg Lysozym.
-
Add double distilled water to Proteinase K. Proteinase K solution should be stored at -20°C.
-
for 50 preparations (# GEN 50 Bac): add 500 µl dd-water to 5 mg Proteinase K.
-
for 250 preparations (# GEN 250 Bac): add 500 µl 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 µl 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 37°C for 1 hour.
-
Centrifuge at 12,000 g for 1 min.
-
Discard the supernatant.
2. Cell Lysis
-
Add 350 µl 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 70°C 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 µl 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 µl 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 µl Washing Buffer (80% Ethanol) into the Spin Column.
-
Centrifuge for 30 sec at 12,000 g.
-
Discard the flow-through.
-
Add 500 µl 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 4°C (for prolonged storage, keep it at -20°C or -80°C).
B. DNA PREPARATION FROM GRAM-NEGATIVE BACTERIA
1. Cell Lysis
-
Harvest 500 µl of cultured bacteria cells by centrifugation at 12,000 g for 1 min.
-
Proceed immediately.
-
Add 350 µl 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 70°C 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 µl 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 µl 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 µl Washing Buffer (80% Ethanol) into the Spin Column.
-
Centrifuge for 30 sec at 12,000 g.
-
Discard the flow-through.
-
Add 500 µl 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 4°C (for prolonged storage, keep it at -20°C or -80°C).
STORAGE
Store at room temperature (15 - 25 °C) for 12 months.
Proteinase K solution and Lysozym solution should be stored at -20°C.
For long term storage place Proteinase K lyophilisate and Lysozym lyophilisate at -20°C.
