Jekyll2019-09-09T17:13:10+00:00http://mwags24.github.io/Prada_Lab_Notebook/feed.xmlLab notebook for Dr. Prada's lab working on Coral phylogeny. Worked on by Myles Wagner.2019-08-14T00:00:00+00:002019-08-14T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/PsbA-NC_PCR10<h2 id="p1-p2-p3-p4-p5-p6-p7-p8-p9-p10-p11-p12-p13-p14">P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14</h2> <h3 id="procedure">Procedure</h3> <p>PsbA-NC is a plastid primer - <a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></p> <p>Samples P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 were used in this PCR (polimerase chain reaction) reaction</p> <p>Two master mixes were made A Master Mix of water, 2x mytaq mix, 1,788F Primer (concentration: 10 micromolar), 2,218R Primer (concentration: 10 micromolar)</p> <p>This Master Mix x4.5</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>1,788F Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>2,218R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 7 tubes For the 2 negative controls of 25 micoleters water no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>The samples were then placed in the thermocycler set to</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>2</td> <td><strong>63</strong></td> <td>1:30</td> <td>1</td> </tr> <tr> <td>3</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>4</td> <td>94°C</td> <td>0:45</td> <td>36</td> </tr> <tr> <td>5</td> <td><strong>63</strong></td> <td>1:00</td> <td>36</td> </tr> <tr> <td>6</td> <td>72°C</td> <td>1:30</td> <td>36</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>8</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 and the two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>P1</td> <td>P2</td> <td>P3</td> <td>Negative Control</td> <td>P4</td> <td>P5</td> <td>P6</td> <td>P14</td> </tr> <tr> <td>Ladder</td> <td>P7</td> <td>P8</td> <td>P9</td> <td>Negative Control</td> <td>P10</td> <td>P11</td> <td>P12</td> <td>P13</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>The Gels have not been run yet for this PCR</p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel7.png" alt="08-14-19_PsbA-NC_Gel7" title="8/14/19 PsbA-NC Gel #7" /></p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel8.png" alt="08-14-19_PsbA-NC_Gel8" title="8/14/19 PsbA-NC Gel #8" /></p> <h3 id="notes">Notes</h3> <p>This PCR was done with the other PCRs from E15-E54 and P1-P16</p> <p>instead of a positive control P14 and P13 were added to the end of these tubes</p>

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14

2019-08-14T00:00:00+00:002019-08-14T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/PsbA-NC_PCR7<h2 id="e15-e16-e17-e18-e19-e20-e21-e22-e23-e24-e25-e26">E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26</h2> <h3 id="procedure">Procedure</h3> <p>PsbA-NC is a plastid primer - <a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></p> <p>Samples E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 were used in this PCR (polimerase chain reaction) reaction</p> <p>Two master mixes were made A Master Mix of water, 2x mytaq mix, 1,788F Primer (concentration: 10 micromolar), 2,218R Primer (concentration: 10 micromolar)</p> <p>This Master Mix x4.5</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>1,788F Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>2,218R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 7 tubes For the 2 negative controls of 25 micoleters water no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>The samples were then placed in the thermocycler set to</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>2</td> <td><strong>63</strong></td> <td>1:30</td> <td>1</td> </tr> <tr> <td>3</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>4</td> <td>94°C</td> <td>0:45</td> <td>36</td> </tr> <tr> <td>5</td> <td><strong>63</strong></td> <td>1:00</td> <td>36</td> </tr> <tr> <td>6</td> <td>72°C</td> <td>1:30</td> <td>36</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>8</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 and the two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E15</td> <td>E16</td> <td>E17</td> <td>Negative Control</td> <td>E18</td> <td>E19</td> <td>E20</td> <td>Positive control - E7</td> </tr> <tr> <td>Ladder</td> <td>E21</td> <td>E22</td> <td>E23</td> <td>Negative Control</td> <td>E24</td> <td>E25</td> <td>E26</td> <td>Positive control - E7</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>The Gels have not been run yet for this PCR</p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel1.png" alt="08-14-19_PsbA-NC_Gel1" title="8/14/19 PsbA-NC Gel #1" /></p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel2.png" alt="08-14-19_PsbA-NC_Gel2" title="8/14/19 PsbA-NC Gel #2" /></p> <h3 id="notes">Notes</h3> <p>This PCR was done with the other PCRs from E15-E54 and P1-P16</p>

E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26

2019-08-14T00:00:00+00:002019-08-14T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/PsbA-NC_PCR8<h2 id="e27-e28-e29-e30-e31-e32-e33-e34-e35-e36-e37-e40-e54-p16">E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 E54 P16</h2> <h3 id="procedure">Procedure</h3> <p>PsbA-NC is a plastid primer - <a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></p> <p>Samples E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 E54 P16 were used in this PCR (polimerase chain reaction) reaction</p> <p>Two master mixes were made A Master Mix of water, 2x mytaq mix, 1,788F Primer (concentration: 10 micromolar), 2,218R Primer (concentration: 10 micromolar)</p> <p>This Master Mix x4.5</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>1,788F Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>2,218R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 7 tubes For the 2 negative controls of 25 micoleters water no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>The samples were then placed in the thermocycler set to</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>2</td> <td><strong>63</strong></td> <td>1:30</td> <td>1</td> </tr> <tr> <td>3</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>4</td> <td>94°C</td> <td>0:45</td> <td>36</td> </tr> <tr> <td>5</td> <td><strong>63</strong></td> <td>1:00</td> <td>36</td> </tr> <tr> <td>6</td> <td>72°C</td> <td>1:30</td> <td>36</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>8</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 E54 P16 and the two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E27</td> <td>E28</td> <td>E29</td> <td>Negative Control</td> <td>E30</td> <td>E31</td> <td>E32</td> <td>E54</td> </tr> <tr> <td>Ladder</td> <td>E33</td> <td>E34</td> <td>E35</td> <td>Negative Control</td> <td>E36</td> <td>E37</td> <td>E40</td> <td>P16</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>The Gels have not been run yet for this PCR</p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel3.png" alt="08-14-19_PsbA-NC_Gel3" title="8/14/19 PsbA-NC Gel #3" /></p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel4.png" alt="08-14-19_PsbA-NC_Gel4" title="8/14/19 PsbA-NC Gel #4" /></p> <h3 id="notes">Notes</h3> <p>This PCR was done with the other PCRs from E15-E54 and P1-P16</p> <p>instead of a positive control E54 and P16 were added to the end of these tubes</p>

E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 E54 P16

2019-08-14T00:00:00+00:002019-08-14T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/PsbA-NC_PCR9<h2 id="e41-e42-e43-e44-e45-e46-e47-e48-e49-e50-e51-e52-e53-p15">E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 P15</h2> <h3 id="procedure">Procedure</h3> <p>PsbA-NC is a plastid primer - <a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></p> <p>Samples E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 P15 were used in this PCR (polimerase chain reaction) reaction</p> <p>Two master mixes were made A Master Mix of water, 2x mytaq mix, 1,788F Primer (concentration: 10 micromolar), 2,218R Primer (concentration: 10 micromolar)</p> <p>This Master Mix x4.5</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>1,788F Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>2,218R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 7 tubes For the 2 negative controls of 25 micoleters water no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>The samples were then placed in the thermocycler set to</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>2</td> <td><strong>63</strong></td> <td>1:30</td> <td>1</td> </tr> <tr> <td>3</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>4</td> <td>94°C</td> <td>0:45</td> <td>36</td> </tr> <tr> <td>5</td> <td><strong>63</strong></td> <td>1:00</td> <td>36</td> </tr> <tr> <td>6</td> <td>72°C</td> <td>1:30</td> <td>36</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>8</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 P15 and the two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E41</td> <td>E42</td> <td>E43</td> <td>Negative Control</td> <td>E44</td> <td>E45</td> <td>E46</td> <td>P15</td> </tr> <tr> <td>Ladder</td> <td>E47</td> <td>E48</td> <td>E49</td> <td>Negative Control</td> <td>E50</td> <td>E51</td> <td>E52</td> <td>E53</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>The Gels have not been run yet for this PCR</p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel5.png" alt="08-14-19_PsbA-NC_Gel5" title="8/14/19 PsbA-NC Gel #5" /></p> <p><img src="/Prada_Lab_Notebook/images/08-14-19_PsbA-NC_Gel6.png" alt="08-14-19_PsbA-NC_Gel6" title="8/14/19 PsbA-NC Gel #6" /></p> <h3 id="notes">Notes</h3> <p>This PCR was done with the other PCRs from E15-E54 and P1-P16</p> <p>instead of a positive control E53 and P15 were added to the end of these tubes</p>

E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 P15

2019-08-13T00:00:00+00:002019-08-13T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/CA6_PCR9<h2 id="e53-e54-p13-p14-p15-p16-e8-e9-e10-e11-e12-e13-e14">E53 E54 P13 P14 P15 P16 E8 E9 E10 E11 E12 E13 E14</h2> <h3 id="procedure">Procedure</h3> <ul> <li><a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></li> </ul> <p>Samples E53 E54 P13 P14 P15 P16 E8 E9 E10 E11 E12 E13 E14 sed in this PCR (polimerase chain reaction) reaction</p> <p>First A Master Mix of water, 2x mytaq mix, CA6.38R Primer (concentration: 10 micromolar), CA6.38L Primer (concentration: 10 micromolar)</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>CA6.38R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>CA6.38L Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added there was no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>This PCR was set to Previous PCRs were showing up smeared and unclear. This could be becuase of too much amplified DNA</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>3:00</td> <td>1</td> </tr> <tr> <td>2</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>3</td> <td>58.2°C</td> <td>1:30</td> <td>1</td> </tr> <tr> <td>4</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>5</td> <td>94°C</td> <td>0:30</td> <td>30</td> </tr> <tr> <td>6</td> <td>58.2°C</td> <td>1:00</td> <td>30</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>0:15</td> <td>30</td> </tr> <tr> <td>8</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>9</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>2g agarose and 100ml 1X TAE buffer were mixed to make a 2% agarose 100ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E53 E54 P13 P14 P15 P16 E8 E9 E10 E11 E12 E13 E14 two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E53</td> <td>E54</td> <td>P13</td> <td>Negative Control</td> <td>P14</td> <td>P15</td> <td>P16</td> <td>Positive control - E7</td> </tr> <tr> <td>Ladder</td> <td>E8</td> <td>E9</td> <td>E10</td> <td>Negative Control</td> <td>E11</td> <td>E12</td> <td>E13</td> <td>E14</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>This is the resulting Gel</p> <p><img src="/Prada_Lab_Notebook/images/08-13-19_CA6_Gel1.png" alt="08-13-19_CA6_Gel1" title="8/13/19 CA6 Gel #1" /> <img src="/Prada_Lab_Notebook/images/08-13-19_CA6_Gel2.png" alt="08-13-19_CA6_Gel2" title="8/13/19 CA6 Gel #2" /></p> <h3 id="notes">Notes</h3> <p>This Gel did not come out as well as some of the others, the PCR may have been messed up in some way.</p>

E53 E54 P13 P14 P15 P16 E8 E9 E10 E11 E12 E13 E14

2019-08-13T00:00:00+00:002019-08-13T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/PsbA-NC_PCR6<h2 id="testing-diluting-primers">Testing Diluting primers</h2> <h3 id="procedure">Procedure</h3> <p>PsbA-NC is a plastid primer - <a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></p> <p>Samples E1 E2 E3 E4 E5 E6 E7 were used in this PCR (polimerase chain reaction) reaction</p> <p>Two master mixes were made A Master Mix of water, 2x mytaq mix, 1,788F Primer (concentration: 1 and 5 micromolar), 2,218R Primer (concentration: 1 and 5 micromolar)</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 7 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>66.15 micoleters</td> </tr> <tr> <td>mytaq mix</td> <td>91.9 micoleters</td> </tr> <tr> <td>1,788F Primer (concentration: 1 micromolar)</td> <td>7.35 micoleters</td> </tr> <tr> <td>2,218R Primer (concentration: 1 micromolar)</td> <td>7.35 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <table> <thead> <tr> <th>Component</th> <th>Volume for 7 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>66.15 micoleters</td> </tr> <tr> <td>mytaq mix</td> <td>91.9 micoleters</td> </tr> <tr> <td>1,788F Primer (concentration: 5 micromolar)</td> <td>7.35 micoleters</td> </tr> <tr> <td>2,218R Primer (concentration: 5 micromolar)</td> <td>7.35 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 7 tubes For the 2 negative controls of 25 micoleters water no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>The samples were then placed in the thermocycler set to</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>2</td> <td><strong>64</strong></td> <td>1:30</td> <td>1</td> </tr> <tr> <td>3</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>4</td> <td>94°C</td> <td>0:45</td> <td>36</td> </tr> <tr> <td>5</td> <td><strong>64</strong></td> <td>1:00</td> <td>36</td> </tr> <tr> <td>6</td> <td>72°C</td> <td>1:30</td> <td>36</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>8</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E1 E2 E3 E4 E5 E6 E7 and the two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E1</td> <td>E2</td> <td>E3</td> <td>Negative Control</td> <td>E4</td> <td>E5</td> <td>E6</td> <td>E7</td> </tr> <tr> <td>Ladder</td> <td>E1</td> <td>E2</td> <td>E3</td> <td>Negative Control</td> <td>E4</td> <td>E5</td> <td>E6</td> <td>E7</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>This is the resulting Gel</p> <p><img src="/Prada_Lab_Notebook/images/08-13-19_PsbA-NC_Gel1.png" alt="08-13-19_PsbA-NC_Gel1" title="8/13/19 PsbA-NC Gel #1" /></p> <p><img src="/Prada_Lab_Notebook/images/08-13-19_PsbA-NC_Gel2.png" alt="08-13-19_PsbA-NC_Gel2" title="8/13/19 PsbA-NC Gel #2" /></p> <h3 id="notes">Notes</h3> <p>this was to try to get rid of the secound band on all the previous PCRs</p>

Testing Diluting primers

2019-08-12T00:00:00+00:002019-08-12T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/CA6_PCR5<h2 id="e15-e16-e17-e18-e19-e20-e21-e22-e23-e24-e25-e26">E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26</h2> <h3 id="procedure">Procedure</h3> <ul> <li><a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></li> </ul> <p>Samples E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 sed in this PCR (polimerase chain reaction) reaction</p> <p>First A Master Mix of water, 2x mytaq mix, CA6.38R Primer (concentration: 10 micromolar), CA6.38L Primer (concentration: 10 micromolar)</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>CA6.38R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>CA6.38L Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added there was no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>This PCR was set to Previous PCRs were showing up smeared and unclear. This could be becuase of too much amplified DNA</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>3:00</td> <td>1</td> </tr> <tr> <td>2</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>3</td> <td>58.2°C</td> <td>1:30</td> <td>1</td> </tr> <tr> <td>4</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>5</td> <td>94°C</td> <td>0:30</td> <td>30</td> </tr> <tr> <td>6</td> <td>58.2°C</td> <td>1:00</td> <td>30</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>0:15</td> <td>30</td> </tr> <tr> <td>8</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>9</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 e two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E15</td> <td>E16</td> <td>E17</td> <td>Negative Control</td> <td>E18</td> <td>E19</td> <td>E20</td> <td>Positive control - E7</td> </tr> <tr> <td>Ladder</td> <td>E21</td> <td>E22</td> <td>E23</td> <td>Negative Control</td> <td>E24</td> <td>E25</td> <td>E26</td> <td>Positive control - E7</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>This is the resulting Gel</p> <p><img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel1.png" alt="08-12-19_CA6_Gel1" title="8/12/19 CA6 Gel #1" /> <img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel2.png" alt="08-12-19_CA6_Gel2" title="8/12/19 CA6 Gel #2" /></p> <h3 id="notes">Notes</h3>

E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26

2019-08-12T00:00:00+00:002019-08-12T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/CA6_PCR6<h2 id="e27-e28-e29-e30-e31-e32-e33-e34-e35-e36-e37-e40">E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40</h2> <h3 id="procedure">Procedure</h3> <ul> <li><a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></li> </ul> <p>Samples E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 sed in this PCR (polimerase chain reaction) reaction</p> <p>First A Master Mix of water, 2x mytaq mix, CA6.38R Primer (concentration: 10 micromolar), CA6.38L Primer (concentration: 10 micromolar)</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>CA6.38R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>CA6.38L Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added there was no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>This PCR was set to Previous PCRs were showing up smeared and unclear. This could be becuase of too much amplified DNA</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>3:00</td> <td>1</td> </tr> <tr> <td>2</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>3</td> <td>58.2°C</td> <td>1:30</td> <td>1</td> </tr> <tr> <td>4</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>5</td> <td>94°C</td> <td>0:30</td> <td>30</td> </tr> <tr> <td>6</td> <td>58.2°C</td> <td>1:00</td> <td>30</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>0:15</td> <td>30</td> </tr> <tr> <td>8</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>9</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40 e two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E27</td> <td>E28</td> <td>E29</td> <td>Negative Control</td> <td>E30</td> <td>E31</td> <td>E32</td> <td>Positive control - E7</td> </tr> <tr> <td>Ladder</td> <td>E33</td> <td>E34</td> <td>E35</td> <td>Negative Control</td> <td>E36</td> <td>E37</td> <td>E40</td> <td>Positive control - E7</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>This is the resulting Gel</p> <p><img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel3.png" alt="08-12-19_CA6_Gel3" title="8/12/19 CA6 Gel #3" /> <img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel4.png" alt="08-12-19_CA6_Gel4" title="8/12/19 CA6 Gel #4" /></p> <h3 id="notes">Notes</h3>

E27 E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E40

2019-08-12T00:00:00+00:002019-08-12T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/CA6_PCR7<h2 id="e41-e42-e43-e44-e45-e46-e47-e48-e49-e50-e51-e52">E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52</h2> <h3 id="procedure">Procedure</h3> <ul> <li><a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></li> </ul> <p>Samples E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 sed in this PCR (polimerase chain reaction) reaction</p> <p>First A Master Mix of water, 2x mytaq mix, CA6.38R Primer (concentration: 10 micromolar), CA6.38L Primer (concentration: 10 micromolar)</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>CA6.38R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>CA6.38L Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added there was no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>This PCR was set to Previous PCRs were showing up smeared and unclear. This could be becuase of too much amplified DNA</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>3:00</td> <td>1</td> </tr> <tr> <td>2</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>3</td> <td>58.2°C</td> <td>1:30</td> <td>1</td> </tr> <tr> <td>4</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>5</td> <td>94°C</td> <td>0:30</td> <td>30</td> </tr> <tr> <td>6</td> <td>58.2°C</td> <td>1:00</td> <td>30</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>0:15</td> <td>30</td> </tr> <tr> <td>8</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>9</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>2g agarose and 100ml 1X TAE buffer were mixed to make a 2% agarose 100ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 e two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>E41</td> <td>E42</td> <td>E43</td> <td>Negative Control</td> <td>E44</td> <td>E45</td> <td>E46</td> <td>Positive control - E7</td> </tr> <tr> <td>Ladder</td> <td>E47</td> <td>E48</td> <td>E49</td> <td>Negative Control</td> <td>E50</td> <td>E51</td> <td>E52</td> <td>Positive control - E7</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>This is the resulting Gel</p> <p><img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel5.png" alt="08-12-19_CA6_Gel5" title="8/12/19 CA6 Gel #5" /> <img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel6.png" alt="08-12-19_CA6_Gel6" title="8/12/19 CA6 Gel #6" /></p> <h3 id="notes">Notes</h3>

E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52

2019-08-12T00:00:00+00:002019-08-12T00:00:00+00:00http://mwags24.github.io/Prada_Lab_Notebook/CA6_PCR8<h2 id="p1-p2-p3-p4-p5-p6-p7-p8-p9-p10-p11-p12">P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12</h2> <h3 id="procedure">Procedure</h3> <ul> <li><a href="/Prada_Lab_Notebook/Pimer_Info/">Primer info</a></li> </ul> <p>Samples P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 sed in this PCR (polimerase chain reaction) reaction</p> <p>First A Master Mix of water, 2x mytaq mix, CA6.38R Primer (concentration: 10 micromolar), CA6.38L Primer (concentration: 10 micromolar)</p> <table> <thead> <tr> <th>Component</th> <th>Volume for 14 reactions(+5% error)</th> </tr> </thead> <tbody> <tr> <td>Water</td> <td>132.3 micoleters</td> </tr> <tr> <td>2x mytaq mix</td> <td>183.75 micoleters</td> </tr> <tr> <td>CA6.38R Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>CA6.38L Primer (concentration: 10 micromolar)</td> <td>14.7 micoleters</td> </tr> <tr> <td>BAS (Borine Serum Albumin)</td> <td>1 microliter</td> </tr> </tbody> </table> <p>24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added there was no positive control was available</p> <p>1 microleter of the DNA samples was added to the coresponding tube</p> <p>This PCR was set to Previous PCRs were showing up smeared and unclear. This could be becuase of too much amplified DNA</p> <table> <thead> <tr> <th>Step</th> <th>Temperature</th> <th>Duration</th> <th>cycles</th> </tr> </thead> <tbody> <tr> <td>1</td> <td>94°C</td> <td>3:00</td> <td>1</td> </tr> <tr> <td>2</td> <td>94°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>3</td> <td>58.2°C</td> <td>1:30</td> <td>1</td> </tr> <tr> <td>4</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>5</td> <td>94°C</td> <td>0:30</td> <td>30</td> </tr> <tr> <td>6</td> <td>58.2°C</td> <td>1:00</td> <td>30</td> </tr> <tr> <td>7</td> <td>72°C</td> <td>0:15</td> <td>30</td> </tr> <tr> <td>8</td> <td>72°C</td> <td>2:00</td> <td>1</td> </tr> <tr> <td>9</td> <td>4°C</td> <td>hold</td> <td>-</td> </tr> </tbody> </table> <hr /> <p>these amplified DNA samples were then tested by making a gel</p> <p>to create the Gel the following steps were taken</p> <p>2g agarose and 100ml 1X TAE buffer were mixed to make a 2% agarose 100ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added</p> <p>the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes</p> <p>once hardened enough Used TAE buffer was poured into the gel box to cover the mold</p> <p>5 microliter aliquots of DNA samples P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12 e two controls were made 1 microliter of 5x loading dye was added to each</p> <p>First 3 microliters of the HyperLadder 100bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order</p> <table> <tbody> <tr> <td>Ladder</td> <td>P1</td> <td>P2</td> <td>P3</td> <td>Negative Control</td> <td>P4</td> <td>P5</td> <td>P6</td> <td>Positive control - E7</td> </tr> <tr> <td>Ladder</td> <td>P7</td> <td>P8</td> <td>P9</td> <td>Negative Control</td> <td>P10</td> <td>P11</td> <td>P12</td> <td>Positive control - E7</td> </tr> </tbody> </table> <p>The voltage was set to 100V and run for 60 minutes</p> <p>This is the resulting Gel</p> <p><img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel7.png" alt="08-12-19_CA6_Gel7" title="8/12/19 CA6 Gel #7" /> <img src="/Prada_Lab_Notebook/images/08-12-19_CA6_Gel8.png" alt="08-12-19_CA6_Gel8" title="8/12/19 CA6 Gel #8" /></p> <h3 id="notes">Notes</h3>

P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 P12