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[GUIDELINES]
<strong>SN Media Recipe</strong><br /><br />1.  Pour a 75% Seawater mixture (ie. for <strong>1L</strong>, add<strong> 750ml White Point Seawater</strong> and <strong>250ml MQ Water</strong>) into a clean, capped bottle. Autoclave using the p18 liquid cyle (NOTE: This should take about 2 hours).<br /><br />2.  Let your 75% Seawater cool to room temperature. (NOTE: To speed up the process, put your 75% Seawater in the cold room). <br /><br />3.  Add nutrients to your cooled 75% Seawater to create the final <strong>SN Media</strong> as detailed below:<br /><br />
<table>
<tbody>
<tr>
<td><strong>Amount 75% Seawater</strong></td>
<td><strong>1000mL (1L)</strong></td>
<td><strong>500 mL</strong></td>
<td><strong>250 mL</strong></td>
<td><strong>100 mL</strong></td>
</tr>
<tr>
<td>EDTA (1g/L)</td>
<td>5.75mL</td>
<td>2.875mL</td>
<td>1.45mL</td>
<td>0.75mL</td>
</tr>
<tr>
<td>K<sub>2</sub>HPO<sub>4</sub> (6.1g/L)</td>
<td>2.6mL</td>
<td>1.3mL</td>
<td>0.65mL</td>
<td>0.325mL</td>
</tr>
<tr>
<td>NaNO<sub>3</sub> (30%)</td>
<td>2.6mL</td>
<td>1.3mL</td>
<td>0.65mL</td>
<td>0.325mL</td>
</tr>
<tr>
<td>Na<sub>2</sub>CO<sub>3</sub> (4g/L)</td>
<td>2.6mL</td>
<td>1.3mL</td>
<td>0.65mL</td>
<td>0.325mL</td>
</tr>
<tr>
<td>CTMM (Trace Metal)</td>
<td>1mL</td>
<td>0.5mL</td>
<td>0.25mL</td>
<td>0.125mL</td>
</tr>
<tr>
<td>Vitamin B12 (10mg/mL)</td>
<td>0.1mL (100μL)</td>
<td>0.05mL (50μL)</td>
<td>0.025mL (25μL)</td>
<td>0.0125mL (12.5μL)</td>
</tr>
</tbody>
</table>
<br />4.  Store the <strong>SN Media</strong> outside of direct UV light (eg. sunlight).<br /><br /><strong><br />SN Media + NH<sub>4</sub> Recipe<br /><br /></strong>1.  Pour a <strong>75% Seawater</strong> mixture (ie. for <strong>1L</strong>, add<strong> 750ml White Point Seawater</strong> and <strong>250ml MQ Water</strong>) into a clean, capped bottle. Autoclave using the p18 liquid cyle (NOTE: This should take about 2 hours).<br /><br />2.  Let your 75% Seawater cool to room temperature. (NOTE: To speed up the process, put your 75% Seawater in the cold room). <br /><br />3.  Add <strong>nutrients</strong> to your cooled 75% Seawater to create the final <strong>SN Media + NH<sub>4</sub></strong> as detailed below:<br /><br />
<table>
<tbody>
<tr>
<td><strong>Amount 75% Seawater</strong></td>
<td><strong>1000mL (1L)</strong></td>
<td><strong>500 mL</strong></td>
<td><strong>250 mL</strong></td>
<td><strong>100 mL</strong></td>
</tr>
<tr>
<td>EDTA (1g/L)</td>
<td>5.75mL</td>
<td>2.875mL</td>
<td>1.45mL</td>
<td>0.75mL</td>
</tr>
<tr>
<td>K<sub>2</sub>HPO<sub>4</sub> (6.1g/L)</td>
<td>2.6mL</td>
<td>1.3mL</td>
<td>0.65mL</td>
<td>0.325mL</td>
</tr>
<tr>
<td>NaNO<sub>3</sub> (30%)</td>
<td>2.6mL</td>
<td>1.3mL</td>
<td>0.65mL</td>
<td>0.325mL</td>
</tr>
<tr>
<td>Na<sub>2</sub>CO<sub>3</sub> (4g/L)</td>
<td>2.6mL</td>
<td>1.3mL</td>
<td>0.65mL</td>
<td>0.325mL</td>
</tr>
<tr>
<td>CTMM (Trace Metal)</td>
<td>1mL</td>
<td>0.5mL</td>
<td>0.25mL</td>
<td>0.125mL</td>
</tr>
<tr>
<td>Vitamin B12 (10mg/mL)</td>
<td>0.1mL (100μL)</td>
<td>0.05mL (50μL)</td>
<td>0.025mL (25μL)</td>
<td>0.0125mL (12.5μL)</td>
</tr>
<tr>
<td>1.6M NH<sub>4</sub>Cl</td>
<td>2.6mL</td>
<td>1.3mL</td>
<td>0.65mL</td>
<td>0.325mL</td>
</tr>
</tbody>
</table>
<br />4.  Store the <strong>SN Media + NH<sub>4</sub></strong> outside of direct UV light (eg. sunlight).<br /><br /><br /><strong>50% AMP / 50% SN Media Recipe<br /><br /></strong>1.  Make <strong>1L Turk's Island Salt Mix</strong> in a clean, capped bottle as per below:<br /><br />
<table>
<tbody>
<tr>
<td><strong>Chemical</strong></td>
<td><strong>g/1000mL (1L)</strong></td>
<td><strong>g/2000ml (2L)</strong></td>
</tr>
<tr>
<td>NaCl</td>
<td>28.11</td>
<td>56.22</td>
</tr>
<tr>
<td>MgSO<sub>4</sub>-7H<sub>2</sub>O</td>
<td>6.9</td>
<td>13.8</td>
</tr>
<tr>
<td>MgCl<sub>2</sub>-6H<sub>2</sub>O</td>
<td>5.49</td>
<td>10.98</td>
</tr>
<tr>
<td>CaCl<sub>2</sub>-2H<sub>2</sub>O</td>
<td>1.47</td>
<td>2.94</td>
</tr>
<tr>
<td>KCl</td>
<td>0.67</td>
<td>1.34</td>
</tr>
<tr>
<td>MQ Water</td>
<td>q. to 1000mL</td>
<td>q. to 2000mL</td>
</tr>
</tbody>
</table>
<br />2.  Autoclave Turk's Island Salt Mix using the p18 liquid cycle (NOTE: This should take about 2 hours.) Let your Turk's Island Salt Mix cool to room temperature. (NOTE: To speed up the process, put your Turk's Island Salt Mix in the cold room).<br /><br />3.  Add the<strong> nutrients, buffers, and trace metals</strong> to your cooled Turk's Island Salt Mix to create your <strong>100% AMP1 Media. </strong>(NOTE: NaHCO<sub>3</sub> needs to be made fresh each time)<br /><br />
<table>
<tbody>
<tr>
<td><strong>Reagent</strong></td>
<td><strong>g/1000mL (1L)</strong></td>
<td><strong>g/2000mL (2L)</strong></td>
</tr>
<tr>
<td style="text-align: center;" colspan="3"><strong>Nutrients</strong></td>
</tr>
<tr>
<td>0.1M NaH<sub>2</sub>PO<sub>4</sub>-H<sub>2</sub>O</td>
<td> 500μL</td>
<td> 1000μl (1mL)</td>
</tr>
<tr>
<td>0.8M (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub></td>
<td> 500μl</td>
<td> 1000μl (1mL)</td>
</tr>
<tr>
<td style="text-align: center;" colspan="3"><strong>Buffers</strong></td>
</tr>
<tr>
<td>0.6M NaHCO<sub>3</sub><br /><sup>(Needs to be made fresh)</sup></td>
<td>10 mL</td>
<td>20 mL</td>
</tr>
<tr>
<td>1M HEPES</td>
<td>1 ml </td>
<td>2 ml</td>
</tr>
<tr>
<td style="text-align: center;" colspan="3"><strong>Trace Metals</strong></td>
</tr>
<tr>
<td style="text-align: left;">Stock Trace Metal Mix<br />(NOT the same CTMM <br />used with SN Media and<br />PRO99 Media)</td>
<td style="text-align: left;">10μl</td>
<td style="text-align: center;">20μl</td>
</tr>
</tbody>
</table>
<br />4.  Mix your 100% AMP1 media with SN Media in a 1:1 ratio (ie. for 1L, mix <strong>500mL 100% AMP1</strong> with <strong>500mL SN Media</strong>) in a clean, autoclaved bottle. (Optional: Autoclave on p13 liquid cycle to ensure sterility of media). This is your <strong>50% AMP / 50% SN Media.</strong> <br /><br />5.  Store your 50% AMP / 50% SN Media outside of direct UV light (eg. sunlight).<br /><br /><br /><strong>Pro99 Media Recipe</strong><br /><br />1.  Pour a <strong>100% SIO, Bigelow or Sargasso Seawater</strong> into a clean, capped bottle. Autoclave using the p18 liquid cycle (NOTE: This should take about 2 hours.)<br /><br />2.  Let your 100% Seawater cool to room temperature. (NOTE: To speed up the process, put your 75% Seawater in the cold room).<br /><br />3. Add nutrients to your cooled 100% Seawater to create the final <strong>Pro99</strong> as detailed below:<strong><strong><br /><br /></strong></strong>
<table>
<tbody>
<tr>
<td><strong>Amout 100% Seawater</strong></td>
<td><strong>1000mL (1L)</strong></td>
<td><strong>500mL</strong></td>
<td><strong>250mL</strong></td>
<td><strong>100mL</strong></td>
</tr>
<tr>
<td>1.6M NH<sub>4</sub>Cl</td>
<td>500μl</td>
<td>250μl</td>
<td>125μl</td>
<td>50μl</td>
</tr>
<tr>
<td>0.1M Na<sub>2</sub>HPO<sub>4</sub></td>
<td>500μl</td>
<td>250μl</td>
<td>125μl</td>
<td>50μl</td>
</tr>
<tr>
<td>CTMM (Trace Metal)</td>
<td>100μl</td>
<td>50μl</td>
<td>25μl</td>
<td>10μl</td>
</tr>
</tbody>
</table>
<br />4.  Store the <strong>Pro99 Media</strong> outside of direct UV light (eg. sunlight).<strong><br /><br /><br />Top Agar and Bottom Agar Recipe<br /></strong><br />Add the following amounts of <strong>low melting point agarose (LMP Agarose)</strong> to your pre-made media (previously made using the aforementioned directions) and autoclave on a p13 liquid cycle or microwave until dissolved.<br /><br />
<table>
<tbody>
<tr>
<td><strong>Media Volume</strong></td>
<td><strong>1000mL (1L)</strong></td>
<td><strong>500mL</strong></td>
</tr>
<tr>
<td>Top Agar</td>
<td>5.5g LMP Agarose</td>
<td>2.75g LMP Agarose</td>
</tr>
<tr>
<td>Bottom Agar</td>
<td>5.0g LMP Agarose</td>
<td>2.50g LMP Agarose</td>
</tr>
</tbody>
</table>

[STEPS]