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Fast-n-Easy Plasmid Mini-Prep Kit | DNA Plasmidial Cellco
Fast-n-Easy Plasmid Mini-Prep Kit
Ficha técnica
| Código | Fast-n-Easy Plasmid Mini-Prep Kit |
| Categoria | DNA Plasmidial |
| Marca | Cellco |
Descrição Geral
Fast-n-Easy Plasmid Mini-Prep Kit | DNA Plasmidial - Cellco
Kit baseado em spin-column para isolamento de DNA plasmidial
For in vitro use only!
Envio:
shipped at ambient temperature
Condições de armazenamento:
store at ambient temperature (except RNAse - store at - 20 ºC)
Validade:
12 months
Descrição:
Fast-n-Easy Plasmid Mini-Prep Kit is designed for isolation of high-purity plasmid or cosmid DNA from bacterial cells for subsequent amplification, sequencing, restriction digests or transformations. The 2-step alkaline lysis procedure and binding 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. Timeconsuming phenol-chloroform extraction or alcohol precipitation is not required.
The Lysis Buffer contains an integrated pH indicator to easily control the optimal pH value for DNA binding. Efficient DNA binding (for Column loading) requires a pH lower than 7.5 that is indicated by a color change of the indicator to bright yellow. The kit can either be used in micro-centrifuges or on vacuum manifolds. It enables the extraction of plasmid DNA up to 10 kb length and yields up to 20 µg DNA per preparation. The eluted high-quality plasmid DNA is ready to use for a variety of down-stream application. For subsequent in vitro translation we recommend to add RNase Inhibitor or the application of an additional spin-column or phenol-chloroform based purification step. This avoids any risk of carry-over contamination with RNase due to the previous neutralization step.
Kit Content:
Lysis Buffer including pH indicator
Neutralization Buffer (before use, add RNase A and store at 4°C)
RNase A (store at -20°C)
Activation Buffer
Washing Buffer (before use, add 96-99% Ethanol as indicated on the bottle)
Elution Buffer
Binding Columns
2 ml Collection Tubes
Additional Materials Required:
96-99 % Ethanol
Microtubes 1.5 ml
Preparation procedure:
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 starting, add the following components to the respective bottles:
- Add the RNase A to the Neutralization Buffer and mix well. Neutralization Buffer containing RNase A should be stored at 4 °C.
- The activity of dissolved RNase A in Neutralization Buffer may decrease after several months and small amounts of RNA may be co-purified. In case RNA is detected after plasmid purification, add additional RNase A to the Neutralization Buffer in order to enhance enzyme activities.
- Add 96-99 % Ethanol (not included in the kit) to the Washing Buffer as indicated on the bottle. Please note that the Ethanol concentration of Washing Buffer may decrease during long term storage resulting in a drop-down of the final DNA yield.
| Buffer | DPK-104XS 10 preps | DPK-104S 50 preps | DPK-104L 250 preps |
| Lysis Buffer | 3.2 ml | 16 ml | 80 ml |
| Neutralization Buffer | 3.2 ml add 0.8 mg RNAse A | 16 ml add 4 mg RNAse A | 80 ml add 5 x 4 mg RNAse A |
| Activation Buffer | 1.2 ml | 6 ml | 30 ml |
| Washing Buffer | add 12 ml Ethanol (final volume 15 mL) | add 55 ml Ethanol (final volume 69 mL) | add 140 ml Ethanol to each bottle (final volume 175 mL) |
| Elution Buffer | 1,2 ml | 6 ml | 30 ml |
1. Cell Harvest and Lysis:
- Harvest the 1 ~ 3 ml of bacterial cell culture by centrifuge.
- Discard the supernatant media.
- Resuspend of cell pellet with remained media by vigorously vortexing or pipetting.
- Add 300 ul of Buffer S1. (Buffer S1 is Cell lysis buffer).
- Inverting the tube immediately 2 ~ 3 times.
Do Not Vortexing and Pipetting.
NOTE: At this time, cell ressuspension with Buffer S1(Lysis Buffer) by vorteing or pipetting can clivage gDNA. The cleaved gDNA affects the plasmid DNA preparation step and can be isolated together with the plasmid DNA. This reduces the efficiency of plasmid DNA prep.
For this reason, mix only by the inverting method after adding Buffer S1.
2. Neutralization
- Add 300 µl of Neutralization Buffer (containing RNase A) to sample and mix gently by inverting the tube 4-6 times (do not vortex!).
- Centrifuge at 10,000 g for 5 min at room temperature in a microcentrifuge.
NOTE: The color of the binding mixture should change to bright yellow indicating a pH lower than 7.5 required for optimal DNA binding. An orange or violet color shows a pH >7.5 and indicates an inefficient DNA adsorption. In this case, it is recommended to adjust the pH of the mixture by addition of a small volume of 3 M sodium acetate, pH 5.0 before proceeding.
2 Protein Precipitation:
- Add 100 μl of Protein Precipitation Solution to the cell lysate.
- Mix the solution well by vortexing for 20 sec.
- Centrifuge at 15,000 g for 3 min. (The precipitated protein will be a tight pellet. If the pellet is not tight, repeat mixing, incubate on ice for 10 minutes, and then centrifuge again.)
3. Column Activation:
- Place a Binding Column into a 2 ml collection tube.
- Add 100 µl of Activation Buffer into the Binding Column.
- Centrifuge at 10,000 g for 30 sec in a micro-centrifuge.
4. Column Loading:
- Apply the supernatant from step 2 into the activated Binding Column by decanting or pipetting.
- Centrifuge at 10,000 g for 30 sec.
- Discard the flow-through from the collection tube.
5. Column Washing:
- Apply 500 µl of Washing Buffer (containing Ethanol) to the Binding Column.
- Centrifuge at 10,000 g for 30 sec and discard the flowthrough.
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 Binding Column.
- Centrifuge at 10,000 g for 30 sec and discard the flowthrough.
- Centrifuge again for 2 min to remove residual Washing Buffer.
6. Elution
- Place the Binding Column into a clean 1.5 ml microtube (not provided in the kit).
- Add 30-50 µl Elution Buffer or dd-water to the center of the column membrane.
- Incubate for 1 min at room temperature.
- Centrifuge at 10,000 g for 1 min to elute DNA.
Ultra-pure plasmid DNA is now ready to use!
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