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[DESCRIPTION]
Preparation of iron chloride resuspension buffer using disodium EDTA dihydrate and magnesium chloride in Tris buffer.

[GUIDELINES]
<strong><strong>Recipe as developed by Seth:<br /></strong></strong>
<table>
<tbody>
<tr>
<td><strong>Reagent (Formula Weight) &nbsp;</strong></td>
<td><strong>Amount&nbsp;</strong></td>
<td><strong>Final Concentration</strong></td>
</tr>
<tr>
<td>Tris-base (FW=121.14)&nbsp;</td>
<td>1.51g</td>
<td>&nbsp;0.125M</td>
</tr>
<tr>
<td>Na<sub>2</sub>-EDTA dihydrate (FW= 372.24) &nbsp;</td>
<td>3.72g&nbsp;</td>
<td>0.1M</td>
</tr>
<tr>
<td>MgCl<sub>2</sub> hexahydrate (FW=203.3)** &nbsp;</td>
<td>4.07g&nbsp;</td>
<td>0.2M</td>
</tr>
<tr>
<td>Ascorbic Acid (FW=176.12)<sup>1</sup> &nbsp;</td>
<td>3.52g&nbsp;</td>
<td>0.2M</td>
</tr>
<tr>
<td>5N NaOH&nbsp;</td>
<td>~4.0ml</td>
<td>&nbsp;to pH 6.5 final</td>
</tr>
<tr>
<td>MilliQ H<sub>2</sub>O</td>
<td>&nbsp;to 100ml</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<sup>1</sup> Oxalic acid can be substituted for ascorbic acid to improve virus infectivity. Oxalic acid dihydrate (FW=126.07), use 2.52g/100ml for 0.2M.&nbsp; See below for testing, but for oxalic acid buffer to stay in solution, use half the amount of MgCl<sub>2</sub>-6H<sub>2</sub>O (i.e., 2.035g/100ml).<br />**Tested recipe using 0.2M MgSO4&middot;7H2O (4.93g/100ml) but still turned cloudy then white after final pH.&nbsp;<strong><br /><br />2X Ascorbic Acid Buffer: </strong>Keep the amount of Tris-base, water and NaOH the same, but increase the amount of EDTA, Mg and ascorbate 2x. Check the pH and add NaOH or HCl to get final pH to 6.5. If increasing 2x, you can use 1 ml for every 2 mg Fe(=1 ml for every 2L seawater precipitated).<strong> <br /><br />Notes:</strong> <br />The original formulation for EDTA-Mg buffer used the chemical Mg-EDTA which is no longer available. The new formulation is now a sodium (Na) salt, and it only contains one Mg ion. For this reason, preparation of the resuspension buffer for iron chloride precipitates should be made from EDTA, disodium salt, and MgCl2. The two most common forms of these chemicals is EDTA-Na2-2H2O (dihydrate) and MgCl26H2O (hexahydrate). <br /><br />When preparing this buffer, keep in mind that EDTA needs a pH above 8.0 to dissolve, and will come out of solution when the pH drops below about 5.0. Ascorbic acid also seems to come out of solution if the pH is very high. The amount of reductant (ascorbic acid or oxalic acid) can vary between 0.125M and 0.25M; this formulation uses 0.2M. Since EDTA is dissolved first, this formulation prepares 0.125M Tris using Tris-base, which allows the EDTA to go into solution more quickly.<br /><br />Recipe tested with diluted amounts of key reagents:<br /><br />
<table>
<tbody>
<tr>
<td style="text-align: center;"><strong>Reagent w/normal amount per</strong><br /><strong>100ml</strong></td>
<td style="text-align: center;"><strong>&frac12; Na<sub>2</sub>-EDTA</strong><br /><strong>(1.86g/100ml)</strong></td>
<td style="text-align: center;"><strong>&frac12; MgCl<sub>2</sub></strong><br /><strong>(2.04g/100ml)</strong></td>
<td style="text-align: center;"><strong>&frac12; Oxalic Acid&middot;2H<sub>2</sub>O</strong><br /><strong>(1.46g/100ml)</strong></td>
</tr>
<tr>
<td>Tris-base 1.51g/100ml</td>
<td>clear; pH 10.79</td>
<td>clear; pH 10.82</td>
<td>clear; pH 10.78</td>
</tr>
<tr>
<td>Na<sub>2</sub>-EDTA 3.72g/100ml</td>
<td>clear</td>
<td>clear</td>
<td>clear</td>
</tr>
<tr>
<td>MgCl<sub>2</sub>&middot;6H<sub>2</sub>O 4.07g/100ml</td>
<td>clear; pH 7.68</td>
<td>clear; pH 4.89</td>
<td>clear; pH 4.59</td>
</tr>
<tr>
<td>5N NaOH</td>
<td>none; pH 7.68</td>
<td>1.25ml; pH 7.23</td>
<td>1.5ml; pH 7.51</td>
</tr>
<tr>
<td>Oxalic acid&middot;2H<sub>2</sub>O 2.52g/100ml</td>
<td>white; pH 1.68</td>
<td>cloudy; pH 3.02</td>
<td>white; pH 3.30</td>
</tr>
<tr>
<td>5N NaOH</td>
<td>6.75ml; cleared ~pH<br />4.5; but turned white<br />at pH 6.5; total 5N<br />NaOH=6.75ml</td>
<td>6.25ml; cleared ~pH<br />4.5; but turned a little<br />cloudy at pH 6.5; total<br />5N NaOH=7.5ml</td>
<td>3.75ml; never cleared;<br />became cloudy then<br />white at pH 6.5; total<br />5N NaOH=5.25ml</td>
</tr>
<tr>
<td>QS to 100ml with H<sub>2</sub>O</td>
<td>pH 6.59; white</td>
<td>pH 6.58; cloudy</td>
<td>pH 6.61; white</td>
</tr>
<tr>
<td>Final results</td>
<td>worst of all after 2hr</td>
<td>looks best after 2hr</td>
<td>intermediate after 2hr</td>
</tr>
</tbody>
</table>
<br />Photo of solutions after final pH:<br /><img src="https://s3.amazonaws.com/pr-journal/dxc2xe.jpg" alt="" /><br />Note the &frac12; MgCl<sub>2</sub> beaker on the left is the most clear but still a little cloudy. The &frac12; Oxalic acid is very cloudy, but can still see the stir bar at the bottom. The &frac12; EDTA has an obvious white precipitate at the bottom that will not go back into solution. This picture is about 2 hr after the final pH and QS to 100ml. Stirring does not make the cloudiness or precipitates go into solution.

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