Isolation of Endotoxin-Free Plasmids Midi Kit is a one-hour plasmid purification kit that is specially made for plasmid purification in a quick and convenient interface. The Kit provides high purity plasmid DNA free of endotoxins, proteins, and other organic compounds, thanks to its unique silica-membrane technology and buffer solution. PCR, restriction enzyme digestion, transformation, and sequencing can all be done with the purified plasmid DNA. It's important to remember that the Kit should be kept at room temperature (15-25°C) and that the only acceptable sample type is bacterial overnight culture with a volume of 5-15 ml.
Storage:
Stored at room temperature (15-25℃)
Components:
Specifications:
| Sample amount | 5-15 ml |
| Features | Fast and convenient workflow enables plasmid DNA purification within 1h. Provides high purity plasmid free from endotoxins, proteins and other organic compounds. |
| Application | The purified plasmid DNA can be used directly for molecular biological experiments such as PCR, restriction enzyme digestion, transformation, and sequencing. |
| Sample type | bacterial overnight culture |
- Column equilibration: Place a Spin Column CP4 in a clean collection tube, and add 500 μl Buffer BL to CP4. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP4 back into the collection tube. (Please use freshly treated spin column).
- Harvest 5-15 ml bacterial cells in a centrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15-25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)
- Re-suspend the bacterial pellet in 500 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.
- Add 500 μl Buffer P2 and mix gently and thoroughly by inverting the tube 6-8 times.
- Add 500 μl Buffer P4 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge.
- Transfer the supernatant from step 5 to the Filtration Columns CS (place Filtration Columns CS in a collection tube) by decanting or pipetting. Centrifuge for 2min at 12,000 rpm (~13,400 × g).
- Add 0.3 volume of isopropanol to the flow-through in step 6, mix by inverting the tube, then transfer the mixture to Spin Columns CP4 in a new collection tube.
- Centrifuge for 1 min at 12,000 rpm (~13,400 × g) at room temperature (15-25°C). Discard the flow-through, put the Spin Columns CP4 back to the collection tube.
- Add 500 μl Buffer PD to Spin Columns CP4, centrifuge for 1 min at 12,000 rpm (~13,400 × g). Discard the flow-through, put the Spin Columns CP4 back to the collection tube.
- Add 600 μl Buffer PW (Ensure that ethanol has been added) to Spin Columns CP4, centrifuge for 1 min at 12,000 rpm (~13,400 × g). Discard the flow-through, put the Spin Columns CP4 back to the collection tube.
- Repeat step 10.
- Put the Spin Columns CP4 back to the collection tube. Centrifuge for 2 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW.
- Place the Spin Column CP4 in a new centrifuge tube. To elute DNA, add 100-300 μl Buffer TB to the center of the Spin Column CP4, incubate for 2 min, and centrifuge for 1 min at 12,000 rpm (~13,400 × g).
Note: No cell clumps should be visible after resuspension of the pellet, otherwise incomplete lysis will lower yield and purity.
Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P4. If there is still white precipitation in the supernatant, please centrifuge again.
Note: Spin Columns CP4 maximum loading volume is 700 µl. If the volume of the sample exceeds 700 µl, successively load aliquots onto the Spin Columns CP4.
Note: After adding the Buffer PW, stand for 2-5 min at room temperature (15-25°C) is helpful to remove the impurities.
Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP4 lid and stay at room temperature for a while to get rid of residual ethanol.
Note: If the volume of eluted buffer is less than 100 μl, it may affect recovery efficiency. Repeat step 13 to increase plasmid recovery efficiency. The pH value of eluted buffer will have some influence in eluting; Buffer TB or distilled water (pH 7.0-8.5) is suggested to elute plasmid DNA. For long-term storage of DNA, eluting in Buffer TB and storing at -20°C is recommended, since DNA stored in water is subject to acid hydrolysis.
