Magni:Protocols/buffering: Difference between revisions

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(New page: {{Magni}} <div style="padding: 10px; width: 720px; border: 5px solid #000000;"> = M9 supplemented medium (liquid) = The final composition of the medium is: M9 salts, 1 mM thiamine hydroch...)
 
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= M9 supplemented medium (liquid) =
= Buffered media =
The final composition of the medium is: M9 salts, 1 mM thiamine hydrochloride, 0.2% casamino acids, 2 mM MgSO4, 0.1 mM CaCl2, 0.4% glycerol.
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.


*For every 1 L of medium, add to a bottle or flask (in the following order) while using a sterile magnetic stirrer:
= LB + phosphate buffer =
**739 ml of autoclaved deionized water (blue tap of the Millipore machine)
*We used sodium phosphate buffer at the final concentration of ??? mM.
**100 ul of CaCl2 1M
*For every 1 L of medium, add all the powder ingredients to 900 ml (instead of 1 L) of deionized water and autoclave.
**200 ml of 5X M9 salts
*Add ??? ml of Na2HPO4
**20 ml of casamino acids 10%
*Add ??? ml of NaH2PO4
**34 ml of thiamine hydrochloride 10 mg/ml
**5 ml of glycerol 80%
**2 ml of MgSO4 1M
*Store in the fridge. NOTE: medium occasionally precipitates when stored for long periods.


== CaCl2 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.
*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.
*Bring to a volume of 100 ml.
*Autoclave in a pyrex bottle.
*Filter-sterilize at 0.2 um.
*Store in the fridge and use at room temperature, near the flame.
*Store in the fridge and use at room temperature, near the flame.


== 5X M9 salts ==
== NaH2PO4 1M ==
*Dissolve 56.4 g of powder in approximately 800 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
*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 1 L.
*Bring to a volume of 100 ml.
*Autoclave in a pyrex bottle.
*Filter-sterilize at 0.2 um.
*Store in the fridge and use at room temperature, near the flame.
*Store in the fridge and use at room temperature, near the flame.


== Casamino acids 10% ==
== PIPES 1M pH 7.0 ==
*Dissolve 10 g of powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer (the solution will not become clear before autoclaving).
*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.
*Bring to a volume of 100 ml.
*Autoclave in a pyrex bottle.
*Filter-sterilize at 0.2 um.
*Store in the fridge and use near the flame.
*Store in the fridge and use at room temperature, near the flame.


== Thiamine hydrochloride 10 mg/ml ==
== MOPS 1M pH 7.0 ==
*Dissolve 0.4 g of powder in exactly 40 ml of Milli-Q water (from the filter of the Millipore machine) in a 50-ml tube.
*Dissolve powder (20.9 g) in approximately 70 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
*Filter-sterilize with a 20-ml syringe and a 0.2 um filter.
*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).
*Do not store! use it when needed to prepare the medium.
*Bring to a volume of 100 ml.
 
*Filter-sterilize at 0.2 um.
== Glycerol 80% ==
*Store in the fridge and use at room temperature, near the flame.
*Mix 80 ml of glycerol with 20 ml of deionized water (blue tap of the Millipore machine).
*Autoclave in a pyrex bottle.
*Store at room temperature and use near the flame.


== MgSO4 1M ==
== HEPES 1M pH 7.2 ==
*Dissolve powder in approximately 80 ml of deionized water (blue tap of the Millipore machine) by using a magnetic stirrer.
*Use a ready-made filter-sterilized solution from Gibco
*Bring to a volume of 100 ml.
*Autoclave in a pyrex bottle.
*Store in the fridge and use at room temperature, near the flame.
*Store in the fridge and use at room temperature, near the flame.


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Revision as of 07:03, 5 May 2013

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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 ??? 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 ??? ml of Na2HPO4
  • Add ??? ml of NaH2PO4

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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