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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= | = 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. | |||
*For every 1 L of medium, add to | = 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. | *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. | ||
* | *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. | ||
== | == NaH2PO4 1M == | ||
*Dissolve | *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 | *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. | *Store in the fridge and use at room temperature, near the flame. | ||
== | == PIPES 1M pH 7.0 == | ||
*Dissolve | *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. | ||
* | *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. | ||
== | == MOPS 1M pH 7.0 == | ||
*Dissolve | *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. | |||
* | |||
*Store at room temperature | |||
== | == 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. | *Store in the fridge and use at room temperature, near the flame. | ||
</div> | </div> |
Revision as of 07:03, 5 May 2013
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.