Miniprep

Materials

  • MX1 buffer
    • RNase
      • MX2 buffer
        • MX3 buffer
          • WN Buffer
            • WS Buffer
              • Elution Buffer (EB)
                • Miniprep columns
                  1. If growing from culture, spin down into a pellet by centrifuging on max speed for ~30 seconds
                  1. Resuspend cell pellet in 200µL MX1+RNase

                  To resuspend the cell pellet, scrape it with the tip of the pipette and simultaneously fluff the mixture.

                  The MX1+RNase should already be prepared in its own bottle, in the fridge.

                  1. Add 250µL MX2, invert several times to mix, incubate at room tempeature for 4-5 min.

                  Do not leave the mixture alone!

                  Err on the side of 4 minutes vs. 5 min.

                  1. Add 350µL MX3 and immediately invert
                  1. Spin down at 13k RPM for 10 min. until precipitate is cleanly pelleted.
                  1. Carefully transfer supernatant to column on vacuum manifold.
                  1. Wash column with 500µL WN Buffer
                  1. Wash with 700µL WS Buffer.
                  1. Spin column at 13k RPM for 2 min into waste collection tube to dry.
                  1. Add 50µL of aliquoted EB, incubate at room temp for 1 min.
                  1. Move column into clean microcentrifuge tube, then centrifuge at 13k RPM for 1 min.
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