ORDERING INFORMATION
|
Catalog # |
Product |
Amount |
Price (EUR) |
| # PLA 50 |
ROVALAB Plasmid Mini-Prep Kit |
50 preparations |
43,50 |
| # PLA 250 |
|
250 preparations |
176,50 |
PRODUCT COMPONENTS
|
# 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 µg DNA per preparation.
FEATURES
- Spin column type
- Binding capacity: 20 µg 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 4°C.
-
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 µl Lysis Buffer and mix gently by inverting the tube 4-6 times (do not vortex!).
-
Add 350 µl 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 µl 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 µl 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 µl 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 -20°C.
RNase A containing Resuspension Buffer should be stored at 4°C.
