ORDERING INFORMATION
|
Catalog # |
Product |
Amount |
Price (EUR) |
| # GEN 50 AP |
Genomic DNA Prep Kit for Animals & Plants |
50 preparations |
|
| # GEN 250 AP |
|
250 preparations |
PRODUCT COMPONENTS
|
# GEN 50 AP |
# GEN 250 AP |
|
|
Animal-Plant Lysis Buffer |
20 ml |
100 ml |
|
|
Binding Buffer |
35 ml |
175 ml |
|
|
Proteinase K (final conc. 10 mg/ml) |
5 mg |
5 x 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 Animal-Plant DNA Preparation Kit is designed for rapid and high purity isolation of genomic DNA from animal cells and plant tissue. 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 Proteinase K. Proteinase K solution should be stored at -20°C.
-
for 50 preparations (# GEN 50 AP): add 500 µl dd-water to 5 mg Proteinase K.
-
for 250 preparations (# GEN 250 AP): 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 AP): 60 ml
-
for 250 preparations (# GEN 250 AP): 300 ml
It is essential to use the correct amount of starting material in order to obtain optimal DNA yield and purity. A maximum amount of 80 mg animal material can generally be processed. We recommend starting with not more than 80 mg fresh or frozen animal tissue. Tissues can be stored at -70°C for several months.
Fresh-freeze tissues in liquid nitrogen and grind it thoroughly with a mortar and pestle. Transfer tissue powder into a 1,5 ml microcentrifuge tube cooled with liquid nitrogen.
1. Cell Lysis
-
Add 350 µl Animal-Plant Lysis Buffer to the tissue powder.
-
Vortex vigorously for 30-60 sec.
-
Add 8 µl (10 mg/ml) Proteinase K solution to the tissue 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 new 1.5 ml microcentrifuge tube.
2. Adding Binding Buffer
-
Add 400 µl Binding Buffer to the tissue lysate.
-
Vortex vigorously for 30-60 sec.
-
Centrifuge for 5 min at 12,000 g at room temperature.
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).
B. DNA PREPARATION FROM PLANT TISSUE
It is essential to use the correct amount of starting material in order to obtain optimal DNA yield and purity. A maximum amount of 80 mg plant material can generally be processed. We recommend starting with not more than 80 mg fresh or frozen plant tissue. Tissues can be stored at -70°C for several months.
Fresh-freeze tissues in liquid nitrogen and grind it thoroughly with a mortar and pestle. Transfer tissue powder into a 1,5 ml microcentrifuge tube cooled with liquid nitrogen.
1. Cell Lysis
-
Add 250 µl Animal-Plant Lysis Buffer to the tissue powder.
-
Vortex vigorously for 30-60 sec.
-
Add 8 µl (10 mg/ml) Proteinase K to the tissue 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 new 1.5 ml microcentrifuge tube.
2. Adding Binding Buffer
-
Add 250 µl Binding Buffer to the tissue lysate.
-
Vortex vigorously for 30-60 sec.
-
Add 8 µl (10 mg/ml) Proteinase K solution to the tissue 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.
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 should be stored at -20°C.
For long term storage place Proteinase K lyophilisate at -20°C.
