Plasmid DNA Miniprep Kit
Introduction:
Cat No: PDK-50/ PDK-100 / PDK-200
Mini Plasmid DNA Isolation and Purification
- Kit offers efficient, fast, convenient and cost-effective purification procedure of low endotoxin plasmid DNA and using mini prep – based columns.
- Purification is achieved without isopropanol precipitation of purified plasmid DNA in approximately 60 minutes.
- Purifies 20 µg of plasmid DNA with A260/A280 >1.7 from a 50–100 ml overnight culture of bacteria, transformed with a high-copy-number plasmid.
- The purified DNA is ready for immediate use in all molecular biology procedures, such as fast and conventional digestion with restriction enzymes, PCR, in vitro transcription, transformation, automated sequencing, and transfection in eukaryotic cells.
Description
Materials Provided:
| Material | 100 Prep | 5 Prep |
| *Resuspension Buffer | 30 ml | 1.5 ml (RNase A added) |
| Rnase A Solution (10 µg/µl) | 300 µl | – |
| Lysis Buffer | 30 ml | 1.5 ml |
| Neutralization Buffer | 30 ml | 1.5 ml |
| DNA Binding Buffer (Brown Bottle) | 40 ml | 2 ml |
| **Wash Buffer | 20 ml (add 80 ml of
96 -100 % ethanol |
1 ml (add 4 ml of 96 –
100 % ethanol |
| Elution Buffer | 20 ml | 1 ml |
| Mini Columns | 100 | 5 |
| Tubes as column inserts | 100 | 5 |
Additional requirements: Microcentrifige Tubes , 96 – 100% Absolute ethanol.
Note: **To Wash Buffer add 96-100% ethanol (In 20 ml of wash buffer + 80 ml ethanol).
*Add Rnase A Solution 300 µl to 30 ml Resuspension Buffer.
Procedure:
- Pipette about 1 ml of coli cells into a 1.5 ml microfuge/Eppendorf tubes. Centrifuge the sample at 10,000 rpm for 2-5 minutes at room temperature.
- Discard the supernatant, and resuspend the cell pellet in 250 µl of Resuspension Buffer containing RNase A . Mix by tapping
- Add 250 µl of Lysis Buffer to the cell (Do not vortex)
- Mix the suspension by gently tapping or by inverting the tube up and down 8-10
- Add 250 µl of Neutralization Buffer and mix the solution thoroughly by inverting the tube up and down 8-10 (Do not vortex).
- Centrifuge at 10,000-14,000 rpm for 10 Discard the pellet and save the supernatant.
- Add 375 µl of DNABinding Buffer to the clear supernatant and
- Load 550-600 µl of the mixture on to the DNA spin column, centrifuge for 1 -2 minutes and discard the flow
Note: You can save the remaining half of the lysate and freeze it at -20°C for future use. If you plan to use all of it now, this will probably double the amount of the DNA yield.
- Wash the DNA spin column with 400 µl of Wash Centrifuge the column for 1-2 minutes. Discard the flow through. Wash one more time.
Note: Add 96-100 % ethanol to the Wash Buffer (In 20 ml of wash buffer + 80 ml ethanol).
- Place the DNA spin column into a clean 5 ml Eppendorf tube. Add 50 µl of Elution Buffer / Nuclease Free water for eluting DNA. Incubate for 2 minutes at RT and centrifuge for 1-2 minute.
- Analyze DNAon an agarose
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