Plasmid spin miniprep kit manual (up to 30 ug)
Plasmid DNA purification protocol
Before you start:
Add RNase A to the resuspension solution and mix (for more complete transfer of RNase A, wash the RNase A tube with a small amount of resuspension solution). To make wash solution concentrate ready add 40 ml of ethanol to 10 ml of wash solution concentrate and mix. Put marks on the lids of vials with a resuspension and washing solution.
Make sure that there is no precipitate in the lysis and neutralising solutions. If precipitate is visible, heat to 50°C in a water bath until the precipitate is completely dissolved.
Do not use more than 30 optical units of cultures for plasmid isolation (eg 15 ml a culture with OD600 equal to 2). If necessary to use more cells for plasmid purification you should proportionately increase the volumes of resuspension, lysis and neutralising solutions, and apply the supernatant several times on the column.
- Centrifuge 2.5-7 ml of a overnight culture of bacteria (10-15 ml for low copy plasmids) at 5,000 rpm (4500g) for 5 minutes (or at 13000 rpm (<10000g) for 1 minute).
- Carefully remove the supernatant. Resuspended the pellet in 250 mkl of resuspension solution and transfer it to a 1.5 ml tube.
- Add 250 uL of the lysis solution to the cell suspension, mix carefully by inverting the tube about 6 times (In case of using 10-15 ml of the culture, the five inverting may not be enough. In this case, the contents of the test tubes must be turned several times rapidly. Do not use vortex for mixing. The solution after this step should not be cloudy, it must be translucent and viscous.)
- Add 350 uL of neutralizing solution, gently mix by inverting the tube 10 times (if using 10-15 ml of the culture, shake the tubes for a few seconds; white flakes are formed in solution after mixing).
- Centrifuge 10 000 - 13 000 rpm for 5 minutes.
- Place the columns into the collection tubes. Transfer the supernanant into the column.
- Centrifuge 10 000 - 13 000 rpm for 30 to 45 seconds. Discard the flow-through and place the column back into the same collection tube. Optional. To completely remove the nuclease, when isolating from end A + strains and removing endotoxins, add 500 uL washing solution with guanidinium chloride (use of this solution may result in a 20% reduction in yield). Centrifuge 10 000 - 13 000 rpm for 60 seconds. Discard flow-through.
- Add into the column 500 mkl of washing solution with ethanol. Centrifuge 10 000 - 13 000 rpm for 30 - 45 seconds. Discard flow-through. Repeat this step one more time.
- After discarding the flow-through, place empty columns into same collection tubes and centrifuge 10 000 to 13 000 rpm for 30 - 45 seconds.
- Place the columns in a new 1.5 ml tubes, add 50-100 uL of the Elution buffer into the center of column filter (If 50 μL of the elution buffer is used, the maximum concentration will be obtained; if 100 μL of the Elution buffer is used, the maximum yield will be obtained), incubate 1 min at room temperature. Centrifuge 10 000 - 13 000 rpm for 1 minute.
Also important! When DNA concentration measuring on a cuvette, a DNA sample should be diluted 10-20 times with TE buffer pH 8.5 or with an elution buffer (do not use water), otherwise the measurements will be inaccurate.