Magni:Protocols/buffering

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Buffered media

While M9 is already buffered by M9 salts, LB medium is not. LB, as well as other media, can be buffered by adding a phosphate buffer or a Good buffer. Phosphate buffer cannot be used when metals are present in the medium, as they will precipitate.

LB + phosphate buffer

  • We used sodium phosphate buffer at the final concentration of 100 mM.
  • For every 1 L of medium, add all the powder ingredients to 900 ml (instead of 1 L) of deionized water and autoclave.
  • Add 57.7 ml of Na2HPO4 1M
  • Add 42.3 ml of NaH2PO4 1M

LB + a Good buffer

  • We use PIPES at 50 or 100 mM, while we use MOPS and HEPES at 50 mM.
  • According to the buffer volume BV that has to be added to the medium, for every 1 L of medium, add all the powder ingredients of LB to 1000-BV ml (instead of 1 L) of deionized water and autoclave.
  • Add BV ml of buffer
  • e.g., to prepare LB + 100 mM of PIPES, add powder ingredients to 900 ml of deionized water, autoclave and finally add 100 ml of PIPES 1M.

Na2HPO4 1M

  • Dissolve powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
  • Bring to a volume of 100 ml.
  • Filter-sterilize at 0.2 um.
  • Store in the fridge and use at room temperature, near the flame.

NaH2PO4 1M

  • Dissolve powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
  • Bring to a volume of 100 ml.
  • Filter-sterilize at 0.2 um.
  • Store in the fridge and use at room temperature, near the flame.

PIPES 1M pH 7.0

  • Dissolve powder (30.2 g) in approximately 70 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
  • Adjust pH to 7.0 with 10N NaOH, powder will not dissolve otherwise! (to do this, dip a previously-washed and 2-point-calibrated pH-meter probe into the solution).
  • Bring to a volume of 100 ml.
  • Filter-sterilize at 0.2 um.
  • Store in the fridge and use at room temperature, near the flame.

MOPS 1M pH 7.0

  • Dissolve powder (20.9 g) in approximately 70 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
  • Adjust pH to 7.0 with 10N NaOH (to do this, dip a previously-washed and 2-point-calibrated pH-meter probe into the solution).
  • Bring to a volume of 100 ml.
  • Filter-sterilize at 0.2 um.
  • Store in the fridge and use at room temperature, near the flame.

HEPES 1M pH 7.2

  • Use a ready-made filter-sterilized solution from Gibco
  • Store in the fridge and use at room temperature, near the flame.
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