Team:TU Darmstadt/Protocols/Miniprep

From 2012.igem.org

Contents

Miniprep

The miniprep is used to purify plasmids.

Procedures

Fermentas kit

MiniPrep steps according to the official Fermentas MiniPrep manual.[1]

Promega kit

MiniPrep steps according to the official Promega MiniPrep manual.[2] The following procedure is performed at room temperature.

  1. Transfer 600μl of bacterial culture grown in LB medium to a 1.5ml microcentrifuge tube.
    • Note: If you wish to process larger volumes of bacterial culture (up to 3.0ml), use the following additional steps:
    1. Centrifuge 1.5 ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge.
    2. Discard the supernatant.
    3. To process a total of 3.0ml of culture, add an additional 1.5 ml of bacterial culture to the same tube. Repeat Steps 1 and 2.
    4. Add 600μl of TE buffer or water to the cell pellet, and resuspend completely.
    5. Proceed to Step 2 of the PureYield™ Plasmid Miniprep System protocol
  2. Add 100μl of Cell Lysis Buffer, and mix by inverting the tube 6 times. The solution should change from opaque to clear blue, indicating complete lysis.
    • Note: Proceed to Step 3 within 2 minutes. Excessive lysis can result in denatured plasmid DNA. If processing a large number of samples, process samples in groups of ten or less. Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.
  3. Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly byinverting the tube.
    • The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization.
  4. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
  5. Transfer the supernatant (~900μl) to a PureYield™ Minicolumn.
    • Do not disturb the cell debris pellet. For maximum yield, transfer the supernatant with a pipette.
  6. Place the minicolumn into a PureYield™ Collection Tube, and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
  7. Discard the flowthrough, and place the minicolumn into the same PureYield™ Collection Tube.
  8. Add 200μl of Endotoxin Removal Wash to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds.
    • It is not necessary to empty the PureYield™ Collection Tube.
  9. Add 400μl of Column Wash Solution to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.
  10. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30μl of Elution Buffer directly to the minicolumn matrix. Let stand for 1 minute at room temperature.
    • Notes:
    1. Nuclease-free water at neutral pH can also be used to elute DNA.
    2. For large plasmids (>10kb), warm the Elution Buffer to 50ºC prior to elution, and increase elution volume to 50μl. Also incubate the column at room temperature (22–25°C) for 5–10 minutes before proceeding to Step 11.
  11. Centrifuge at maximum speed in a microcentrifuge for 15 seconds to elute the plasmid DNA. Cap the microcentrifuge tube, and store eluted plasmid DNA at –20°C.

Qiagen kit

MiniPrep steps according to the official Quiagen MiniPrep manual.[3]

  1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
    • Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
    • If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
  2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
    • 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 becomesviscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
    • If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension.
    • If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
  3. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
    • To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
    • If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
  4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
  5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
  6. Centrifuge for 30–60 s. Discard the flow-through.
  7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
    • This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. #* Host strainssuch as XL-1 Blue and DH5α™ do not require this additional wash step.
  8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
  9. Discard the flow-through, and centrifuge for an additional 1 min to remove residualwash buffer.
    • Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
  10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

References

[1]

[2] http://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/101/pureyield%20plasmid%20miniprep%20system%20protocol.pdf?la=en

[3] http://www.qiagen.com/literature/render.aspx?id=370

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