Team:Duke/Notebook/Test

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<h2>9/4/14</h2>
<h2>9/4/14</h2>
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<p>Objective: test backbone shuffled antitracr bacteria</p>
<p>Objective: test backbone shuffled antitracr bacteria</p>
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<ul><li>Started colonies from plates and diluted in to +/- aTc this morning. Let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS for flow.</li><li>Took data to flowjo and plotted histograms below. This is ungated fluorescence</li><li></li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 482.67px;"><img alt="" src="images/image44.jpg" style="width: 624.00px; height: 482.67px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>table with mean GFPAs</li></ul><a href="#" name="096a26566c8f532b5e11f6f110d57e6edec006da"></a><a href="#" name="3"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p></p></td><td><p></p></td><td><p></p></td><td><p>ungated GFPA</p></td><td><p>+/- aTc</p></td></tr><tr><td><p>1: if142014-09-04.001.fcs</p></td><td><p>AC-1</p></td><td><p>+ aTc</p></td><td><p>0.402</p></td><td><p>1.185840708</p></td></tr><tr><td><p>2: if142014-09-04.002.fcs</p></td><td><p>AC-2</p></td><td><p>+ aTc</p></td><td><p>0.455</p></td><td><p>1.197368421</p></td></tr><tr><td><p>3: if142014-09-04.003.fcs</p></td><td><p>G-1</p></td><td><p>+ aTc</p></td><td><p>14.9</p></td><td><p>0.949044586</p></td></tr><tr><td><p>4: if142014-09-04.004.fcs</p></td><td><p>G-2</p></td><td><p>+ aTc</p></td><td><p>11.7</p></td><td><p>0.573529412</p></td></tr><tr><td><p>5: if142014-09-04.005.fcs</p></td><td><p>G-3</p></td><td><p>+ aTc</p></td><td><p>8.91</p></td><td><p>0.530357143</p></td></tr><tr><td><p>6: if142014-09-04.006.fcs</p></td><td><p>G-4</p></td><td><p>+ aTc</p></td><td><p>5.92</p></td><td><p>0.34619883</p></td></tr><tr><td><p>7: if142014-09-04.007.fcs</p></td><td><p>C-1</p></td><td><p>+ aTc</p></td><td><p>186</p></td><td><p>1.016393443</p></td></tr><tr><td><p>8: if142014-09-04.008.fcs</p></td><td><p>C-2</p></td><td><p>+ aTc</p></td><td><p>224</p></td><td><p>1.230769231</p></td></tr><tr><td><p>9: if142014-09-04.009.fcs</p></td><td><p>C-3</p></td><td><p>+ aTc</p></td><td><p>214</p></td><td><p>0.922413793</p></td></tr><tr><td><p>10: if142014-09-04.010.fcs</p></td><td><p>C-4</p></td><td><p>+ aTc</p></td><td><p>222</p></td><td><p>1.193548387</p></td></tr><tr><td><p>11: if142014-09-04.011.fcs</p></td><td><p>AC-1</p></td><td><p>- aTc</p></td><td><p>0.339</p></td><td><p></p></td></tr><tr><td><p>12: if142014-09-04.012.fcs</p></td><td><p>AC-2</p></td><td><p>- aTc</p></td><td><p>0.38</p></td><td><p></p></td></tr><tr><td><p>13: if142014-09-04.013.fcs</p></td><td><p>G-1</p></td><td><p>- aTc</p></td><td><p>15.7</p></td><td><p></p></td></tr><tr><td><p>14: if142014-09-04.014.fcs</p></td><td><p>G-2</p></td><td><p>- aTc</p></td><td><p>20.4</p></td><td><p></p></td></tr><tr><td><p>15: if142014-09-04.015.fcs</p></td><td><p>G-3</p></td><td><p>- aTc</p></td><td><p>16.8</p></td><td><p></p></td></tr><tr><td><p>16: if142014-09-04.016.fcs</p></td><td><p>G-4</p></td><td><p>- aTc</p></td><td><p>17.1</p></td><td><p></p></td></tr><tr><td><p>17: if142014-09-04.017.fcs</p></td><td><p>C-1</p></td><td><p>- aTc</p></td><td><p>183</p></td><td><p></p></td></tr><tr><td><p>18: if142014-09-04.018.fcs</p></td><td><p>C-2</p></td><td><p>- aTc</p></td><td><p>182</p></td><td><p></p></td></tr><tr><td><p>19: if142014-09-04.019.fcs</p></td><td><p>C-3</p></td><td><p>- aTc</p></td><td><p>232</p></td><td><p></p></td></tr><tr><td><p>20: if142014-09-04.020.fcs</p></td><td><p>C-4</p></td><td><p>- aTc</p></td><td><p>186</p></td><td><p></p></td></tr></tbody></table><ul><li>aTc treatment seems to be moving fluor in the opposite direction of what we&rsquo;d expect of antitracr was doing its job. The degree is inconsistent, reflow is called for. </li></ul><p></p><p></p><p></p><p></p><hr style="page-break-before:always;display:none;"><p></p><hr style="page-break-before:always;display:none;"><p></p><p></p><h2><a name="h.ohhk3mdbkmcm"></a>9/8/14</h2><p>Objective: retest backbone shuffled antitracr bacteria</p><ul><li>Yesterday picked 4 new colonies from plates and 4 from 9/4/14 frozen stocks and grew in LB + antibiotics overnight. Diluted in to +/- aTc this morning and let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS</li><li>G1-4 are old frozen coli with K608012, pdCas9-GFP1, and 1A2-R0040-antitracr, C1-4 are old frozen with pdCas9. clones 5-8 are new from plates. AC are no-fluor (1A2 and pdCas9) but they were diluted in to triple antibiotic medium instead of double so they did not grow and are not reliable indicators of bg fluorescence on this run.</li><li>After flow, isolated singlets with flowjo (smalls) or left alone (bugs) and got gfpa unmodified or normalized by fsca or ssca. </li><li></li></ul><a href="#" name="777f62c089f7173b14ec6d88236db6c3e53afff8"></a><a href="#" name="4"></a><table cellpadding="0" cellspacing="0"<><tbody><tr><td><p></p></td><td><p></p></td><td><p></p></td><td><p></p></td><td><p>smalls</p></td><td><p></p></td><td><p></p></td><td><p>bugs</p></td><td><p></p></td></tr><tr><td><p></p></td><td><p></p></td><td><p>aTc</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td></tr><tr><td><p>1: if142014-09-08.001.fcs</p></td><td><p>G1</p></td><td><p>+</p></td><td><p>5.25</p></td><td><p>243</p></td><td><p>112</p></td><td><p>7.79</p></td><td><p>247</p></td><td><p>114</p></td></tr><tr><td><p>2: if142014-09-08.002.fcs</p></td><td><p>G2</p></td><td><p>+</p></td><td><p>4.41</p></td><td><p>226</p></td><td><p>78.1</p></td><td><p>7.12</p></td><td><p>228</p></td><td><p>76.2</p></td></tr><tr><td><p>3: if142014-09-08.003.fcs</p></td><td><p>G3</p></td><td><p>+</p></td><td><p>4.66</p></td><td><p>231</p></td><td><p>84.8</p></td><td><p>7.8</p></td><td><p>231</p></td><td><p>83.4</p></td></tr><tr><td><p>4: if142014-09-08.004.fcs</p></td><td><p>G4</p></td><td><p>+</p></td><td><p>3.86</p></td><td><p>191</p></td><td><p>70.1</p></td><td><p>5.99</p></td><td><p>192</p></td><td><p>67.9</p></td></tr><tr><td><p>5: if142014-09-08.005.fcs</p></td><td><p>G1</p></td><td><p>-</p></td><td><p>6.81</p></td><td><p>311</p></td><td><p>145</p></td><td><p>8.56</p></td><td><p>310</p></td><td><p>143</p></td></tr><tr><td><p>6: if142014-09-08.006.fcs</p></td><td><p>G2</p></td><td><p>-</p></td><td><p>6.37</p></td><td><p>337</p></td><td><p>153</p></td><td><p>7.25</p></td><td><p>323</p></td><td><p>145</p></td></tr><tr><td><p>7: if142014-09-08.007.fcs</p></td><td><p>G3</p></td><td><p>-</p></td><td><p>6.5</p></td><td><p>318</p></td><td><p>143</p></td><td><p>8.24</p></td><td><p>305</p></td><td><p>136</p></td></tr><tr><td><p>8: if142014-09-08.008.fcs</p></td><td><p>G4</p></td><td><p>-</p></td><td><p>6.14</p></td><td><p>298</p></td><td><p>140</p></td><td><p>7.66</p></td><td><p>288</p></td><td><p>131</p></td></tr><tr><td><p>9: if142014-09-08.009.fcs</p></td><td><p>C1</p></td><td><p>+</p></td><td><p>88.9</p></td><td><p>4071</p></td><td><p>1777</p></td><td><p>117</p></td><td><p>3978</p></td><td><p>1706</p></td></tr><tr><td><p>10: if142014-09-08.010.fcs</p></td><td><p>C2</p></td><td><p>+</p></td><td><p>83.5</p></td><td><p>4073</p></td><td><p>1547</p></td><td><p>120</p></td><td><p>3969</p></td><td><p>1422</p></td></tr><tr><td><p>11: if142014-09-08.011.fcs</p></td><td><p>C3</p></td><td><p>+</p></td><td><p>81.7</p></td><td><p>3957</p></td><td><p>1551</p></td><td><p>115</p></td><td><p>3840</p></td><td><p>1437</p></td></tr><tr><td><p>12: if142014-09-08.012.fcs</p></td><td><p>C4</p></td><td><p>+</p></td><td><p>77.2</p></td><td><p>3915</p></td><td><p>1577</p></td><td><p>95.7</p></td><td><p>3883</p></td><td><p>1503</p></td></tr><tr><td><p>13: if142014-09-08.013.fcs</p></td><td><p>C1</p></td><td><p>-</p></td><td><p>77.7</p></td><td><p>3686</p></td><td><p>1471</p></td><td><p>100</p></td><td><p>3545</p></td><td><p>1411</p></td></tr><tr><td><p>14: if142014-09-08.014.fcs</p></td><td><p>C2</p></td><td><p>-</p></td><td><p>64.5</p></td><td><p>3093</p></td><td><p>1445</p></td><td><p>88.3</p></td><td><p>2962</p></td><td><p>1373</p></td></tr><tr><td><p>15: if142014-09-08.015.fcs</p></td><td><p>C3</p></td><td><p>-</p></td><td><p>80.9</p></td><td><p>3971</p></td><td><p>1541</p></td><td><p>106</p></td><td><p>3771</p></td><td><p>1432</p></td></tr><tr><td><p>16: if142014-09-08.016.fcs</p></td><td><p>C4</p></td><td><p>-</p></td><td><p>58.9</p></td><td><p>2899</p></td><td><p>1299</p></td><td><p>79.7</p></td><td><p>2727</p></td><td><p>1220</p></td></tr><tr><td><p>17: if142014-09-08.017.fcs</p></td><td><p>G5</p></td><td><p>+</p></td><td><p>4.1</p></td><td><p>210</p></td><td><p>79.1</p></td><td><p>5.4</p></td><td><p>209</p></td><td><p>76.2</p></td></tr><tr><td><p>18: if142014-09-08.018.fcs</p></td><td><p>G6</p></td><td><p>+</p></td><td><p>4.9</p></td><td><p>225</p></td><td><p>98.2</p></td><td><p>7.07</p></td><td><p>226</p></td><td><p>97.6</p></td></tr><tr><td><p>19: if142014-09-08.019.fcs</p></td><td><p>G7</p></td><td><p>+</p></td><td><p>4.42</p></td><td><p>220</p></td><td><p>84.7</p></td><td><p>5.97</p></td><td><p>218</p></td><td><p>81.7</p></td></tr><tr><td><p>20: if142014-09-08.020.fcs</p></td><td><p>G8</p></td><td><p>+</p></td><td><p>4.19</p></td><td><p>211</p></td><td><p>81.3</p></td><td><p>5.68</p></td><td><p>213</p></td><td><p>79.5</p></td></tr><tr><td><p>21: if142014-09-08.021.fcs</p></td><td><p>G5</p></td><td><p>-</p></td><td><p>9.73</p></td><td><p>477</p></td><td><p>201</p></td><td><p>12.5</p></td><td><p>455</p></td><td><p>190</p></td></tr><tr><td><p>22: if142014-09-08.022.fcs</p></td><td><p>G6</p></td><td><p>-</p></td><td><p>9.68</p></td><td><p>450</p></td><td><p>197</p></td><td><p>12.9</p></td><td><p>449</p></td><td><p>196</p></td></tr><tr><td><p>23: if142014-09-08.023.fcs</p></td><td><p>G7</p></td><td><p>-</p></td><td><p>10.1</p></td><td><p>482</p></td><td><p>212</p></td><td><p>13.3</p></td><td><p>464</p></td><td><p>198</p></td></tr><tr><td><p>24: if142014-09-08.024.fcs</p></td><td><p>G8</p></td><td><p>-</p></td><td><p>10.1</p></td><td><p>489</p></td><td><p>216</p></td><td><p>13.3</p></td><td><p>473</p></td><td><p>204</p></td></tr><tr><td><p>25: if142014-09-08.025.fcs</p></td><td><p>C5</p></td><td><p>+</p></td><td><p>79.6</p></td><td><p>3958</p></td><td><p>1486</p></td><td><p>113</p></td><td><p>3813</p></td><td><p>1374</p></td></tr><tr><td><p>26: if142014-09-08.026.fcs</p></td><td><p>C6</p></td><td><p>+</p></td><td><p>80.8</p></td><td><p>4045</p></td><td><p>1514</p></td><td><p>113</p></td><td><p>4043</p></td><td><p>1425</p></td></tr><tr><td><p>27: if142014-09-08.027.fcs</p></td><td><p>C7</p></td><td><p>+</p></td><td><p>80.7</p></td><td><p>4053</p></td><td><p>1491</p></td><td><p>116</p></td><td><p>3921</p></td><td><p>1375</p></td></tr><tr><td><p>28: if142014-09-08.028.fcs</p></td><td><p>C8</p></td><td><p>+</p></td><td><p>79.8</p></td><td><p>3804</p></td><td><p>1555</p></td><td><p>103</p></td><td><p>3634</p></td><td><p>1508</p></td></tr><tr><td><p>29: if142014-09-08.029.fcs</p></td><td><p>C5</p></td><td><p>-</p></td><td><p>68.9</p></td><td><p>3268</p></td><td><p>1505</p></td><td><p>95.7</p></td><td><p>3128</p></td><td><p>1419</p></td></tr><tr><td><p>30: if142014-09-08.030.fcs</p></td><td><p>C6</p></td><td><p>-</p></td><td><p>80.2</p></td><td><p>3806</p></td><td><p>1639</p></td><td><p>110</p></td><td><p>3630</p></td><td><p>1541</p></td></tr><tr><td><p>31: if142014-09-08.031.fcs</p></td><td><p>C7</p></td><td><p>-</p></td><td><p>67.8</p></td><td><p>3240</p></td><td><p>1498</p></td><td><p>91.7</p></td><td><p>3041</p></td><td><p>1412</p></td></tr><tr><td><p>32: if142014-09-08.032.fcs</p></td><td><p>C8</p></td><td><p>-</p></td><td><p>83.7</p></td><td><p>3810</p></td><td><p>1675</p></td><td><p>107</p></td><td><p>3618</p></td><td><p>1621</p></td></tr><tr><td><p>33: if142014-09-08.033.fcs</p></td><td><p>AC1</p></td><td><p>+</p></td><td><p>0.895</p></td><td><p>40.2</p></td><td><p>16.8</p></td><td><p>0.854</p></td><td><p>34.9</p></td><td><p>12.9</p></td></tr><tr><td><p>34: if142014-09-08.034.fcs</p></td><td><p>AC3</p></td><td><p>+</p></td><td><p>0.539</p></td><td><p>28.4</p></td><td><p>11</p></td><td><p>0.785</p></td><td><p>27.4</p></td><td><p>10.7</p></td></tr><tr><td><p>35: if142014-09-08.035.fcs</p></td><td><p>AC1</p></td><td><p>-</p></td><td><p>0.464</p></td><td><p>25.8</p></td><td><p>9.39</p></td><td><p>0.605</p></td><td><p>25.9</p></td><td><p>8.65</p></td></tr><tr><td><p>36: if142014-09-08.036.fcs</p></td><td><p>AC3</p></td><td><p>-</p></td><td><p>0.532</p></td><td><p>28.3</p></td><td><p>10.4</p></td><td><p>0.661</p></td><td><p>26.2</p></td><td><p>9.23</p></td></tr></tbody></table><p></p><ul><li>Got ratios of fluorescence in +/- aTc and am reporting them in the table below</li></ul><a href="#" name="803fb1152dc946dc3b98f720d1637f36481109e8"></a><a href="#" name="5"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>n/a</p></td><td><p>n/a</p></td><td><p>+/-</p></td><td><p>smalls</p></td><td><p></p></td><td><p></p></td><td><p>bugs</p></td><td><p></p></td></tr><tr><td><p>n/a</p></td><td><p>n/a</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td></tr><tr><td><p>1: if142014-09-08.001.fcs</p></td><td><p>G1</p></td><td><p>0.771</p></td><td><p>0.781</p></td><td><p>0.772</p></td><td><p>0.910</p></td><td><p>0.797</p></td><td><p>0.797</p></td></tr><tr><td><p>2: if142014-09-08.002.fcs</p></td><td><p>G2</p></td><td><p>0.692</p></td><td><p>0.671</p></td><td><p>0.510</p></td><td><p>0.982</p></td><td><p>0.706</p></td><td><p>0.526</p></td></tr><tr><td><p>3: if142014-09-08.003.fcs</p></td><td><p>G3</p></td><td><p>0.717</p></td><td><p>0.726</p></td><td><p>0.593</p></td><td><p>0.947</p></td><td><p>0.757</p></td><td><p>0.613</p></td></tr><tr><td><p>4: if142014-09-08.004.fcs</p></td><td><p>G4</p></td><td><p>0.629</p></td><td><p>0.641</p></td><td><p>0.501</p></td><td><p>0.782</p></td><td><p>0.667</p></td><td><p>0.518</p></td></tr><tr><td><p>9: if142014-09-08.009.fcs</p></td><td><p>C1</p></td><td><p>1.144</p></td><td><p>1.104</p></td><td><p>1.208</p></td><td><p>1.170</p></td><td><p>1.122</p></td><td><p>1.209</p></td></tr><tr><td><p>10: if142014-09-08.010.fcs</p></td><td><p>C2</p></td><td><p>1.295</p></td><td><p>1.317</p></td><td><p>1.071</p></td><td><p>1.359</p></td><td><p>1.340</p></td><td><p>1.036</p></td></tr><tr><td><p>11: if142014-09-08.011.fcs</p></td><td><p>C3</p></td><td><p>1.010</p></td><td><p>0.996</p></td><td><p>1.006</p></td><td><p>1.085</p></td><td><p>1.018</p></td><td><p>1.003</p></td></tr><tr><td><p>12: if142014-09-08.012.fcs</p></td><td><p>C4</p></td><td><p>1.311</p></td><td><p>1.350</p></td><td><p>1.214</p></td><td><p>1.201</p></td><td><p>1.424</p></td><td><p>1.232</p></td></tr><tr><td><p>17: if142014-09-08.017.fcs</p></td><td><p>G5</p></td><td><p>0.421</p></td><td><p>0.440</p></td><td><p>0.394</p></td><td><p>0.432</p></td><td><p>0.459</p></td><td><p>0.401</p></td></tr><tr><td><p>18: if142014-09-08.018.fcs</p></td><td><p>G6</p></td><td><p>0.506</p></td><td><p>0.500</p></td><td><p>0.498</p></td><td><p>0.548</p></td><td><p>0.503</p></td><td><p>0.498</p></td></tr><tr><td><p>19: if142014-09-08.019.fcs</p></td><td><p>G7</p></td><td><p>0.438</p></td><td><p>0.456</p></td><td><p>0.400</p></td><td><p>0.449</p></td><td><p>0.470</p></td><td><p>0.413</p></td></tr><tr><td><p>20: if142014-09-08.020.fcs</p></td><td><p>G8</p></td><td><p>0.415</p></td><td><p>0.431</p></td><td><p>0.376</p></td><td><p>0.427</p></td><td><p>0.450</p></td><td><p>0.390</p></td></tr><tr><td><p>25: if142014-09-08.025.fcs</p></td><td><p>C5</p></td><td><p>1.155</p></td><td><p>1.211</p></td><td><p>0.987</p></td><td><p>1.181</p></td><td><p>1.219</p></td><td><p>0.968</p></td></tr><tr><td><p>26: if142014-09-08.026.fcs</p></td><td><p>C6</p></td><td><p>1.007</p></td><td><p>1.063</p></td><td><p>0.924</p></td><td><p>1.027</p></td><td><p>1.114</p></td><td><p>0.925</p></td></tr><tr><td><p>27: if142014-09-08.027.fcs</p></td><td><p>C7</p></td><td><p>1.190</p></td><td><p>1.251</p></td><td><p>0.995</p></td><td><p>1.265</p></td><td><p>1.289</p></td><td><p>0.974</p></td></tr><tr><td><p>28: if142014-09-08.028.fcs</p></td><td><p>C8</p></td><td><p>0.953</p></td><td><p>0.998</p></td><td><p>0.928</p></td><td><p>0.963</p></td><td><p>1.004</p></td><td><p>0.930</p></td></tr><tr><td><p>33: if142014-09-08.033.fcs</p></td><td><p>AC1</p></td><td><p>1.929</p></td><td><p>1.558</p></td><td><p>1.789</p></td><td><p>1.412</p></td><td><p>1.347</p></td><td><p>1.491</p></td></tr><tr><td><p>34: if142014-09-08.034.fcs</p></td><td><p>AC3</p></td><td><p>1.013</p></td><td><p>1.004</p></td><td><p>1.058</p></td><td><p>1.188</p></td><td><p>1.046</p></td><td><p>1.159</p></td></tr></tbody></table><ul><li></li></ul><hr style="page-break-before:always;display:none;"><p><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 560.00px; height: 420.00px;"><img alt="" src="images/image19.jpg" style="width: 560.00px; height: 420.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></p><p>Box plot of just G1-4 and C1-4. Note that the G clones from plates (clones 5-8) have even smaller ratios. </p><hr style="page-break-before:always;display:none;"><p></p><p></p><p></p><p></p><hr style="page-break-before:always;display:none;"><p></p><p></p><p></p><p></p><h2><a name="h.h7pf932a50dt"></a>9/9/14</h2><p>Objective: assembly decoy binding site arrays</p><ul><li>Garima sets up a PCR and is asked to fill in notes about it.</li><li>Results</li><li>Charlie sets up a new set of tubes for PCR with a 5 rx master mix containing</li></ul><ul><li>190uL H2O</li><li>50 uL Q5 buffer</li><li>2.0 uL oligo 1 (gfp1-spacer-gfp1)</li><li>2.5 uL Q5 poly</li><li>5 uL dNTPs</li></ul><ul><li>And then divides that in to four tubes before adding in the following volumes of oligo 2 (spacer-gfp1-space)</li></ul><a href="#" name="fd71260aa00de3f043dfc426450b7d96e13e45b0"></a><a href="#" name="6"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>tube number</p></td><td><p>1</p></td><td><p>2</p></td><td><p>3</p></td><td><p>4</p></td></tr><tr><td><p>uL oligo 2</p></td><td><p>0</p></td><td><p>0.5</p></td><td><p>1.0</p></td><td><p>2.0</p></td></tr></tbody></table><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 10, 2014 1:24:31 PM.jpg" src="images/image04.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><ul><li>Streakiness on the row with 0.5uL of oligo 2 looks promising? IGEM protocol on the thermal cycler modified</li></ul><h2><a name="h.3838tuwxw3ds"></a>
+
<ul><li>Started colonies from plates and diluted in to +/- aTc this morning. Let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS for flow.</li><li>Took data to flowjo and plotted histograms below. This is ungated fluorescence</li><li></li><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 482.67px;"><img alt="" src="https://static.igem.org/mediawiki/2014/9/90/9-4-14-1.jpg" style="width: 624.00px; height: 482.67px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li><li>table with mean GFPAs</li></ul><a href="#" name="096a26566c8f532b5e11f6f110d57e6edec006da"></a><a href="#" name="3"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p></p></td><td><p></p></td><td><p></p></td><td><p>ungated GFPA</p></td><td><p>+/- aTc</p></td></tr><tr><td><p>1: if142014-09-04.001.fcs</p></td><td><p>AC-1</p></td><td><p>+ aTc</p></td><td><p>0.402</p></td><td><p>1.185840708</p></td></tr><tr><td><p>2: if142014-09-04.002.fcs</p></td><td><p>AC-2</p></td><td><p>+ aTc</p></td><td><p>0.455</p></td><td><p>1.197368421</p></td></tr><tr><td><p>3: if142014-09-04.003.fcs</p></td><td><p>G-1</p></td><td><p>+ aTc</p></td><td><p>14.9</p></td><td><p>0.949044586</p></td></tr><tr><td><p>4: if142014-09-04.004.fcs</p></td><td><p>G-2</p></td><td><p>+ aTc</p></td><td><p>11.7</p></td><td><p>0.573529412</p></td></tr><tr><td><p>5: if142014-09-04.005.fcs</p></td><td><p>G-3</p></td><td><p>+ aTc</p></td><td><p>8.91</p></td><td><p>0.530357143</p></td></tr><tr><td><p>6: if142014-09-04.006.fcs</p></td><td><p>G-4</p></td><td><p>+ aTc</p></td><td><p>5.92</p></td><td><p>0.34619883</p></td></tr><tr><td><p>7: if142014-09-04.007.fcs</p></td><td><p>C-1</p></td><td><p>+ aTc</p></td><td><p>186</p></td><td><p>1.016393443</p></td></tr><tr><td><p>8: if142014-09-04.008.fcs</p></td><td><p>C-2</p></td><td><p>+ aTc</p></td><td><p>224</p></td><td><p>1.230769231</p></td></tr><tr><td><p>9: if142014-09-04.009.fcs</p></td><td><p>C-3</p></td><td><p>+ aTc</p></td><td><p>214</p></td><td><p>0.922413793</p></td></tr><tr><td><p>10: if142014-09-04.010.fcs</p></td><td><p>C-4</p></td><td><p>+ aTc</p></td><td><p>222</p></td><td><p>1.193548387</p></td></tr><tr><td><p>11: if142014-09-04.011.fcs</p></td><td><p>AC-1</p></td><td><p>- aTc</p></td><td><p>0.339</p></td><td><p></p></td></tr><tr><td><p>12: if142014-09-04.012.fcs</p></td><td><p>AC-2</p></td><td><p>- aTc</p></td><td><p>0.38</p></td><td><p></p></td></tr><tr><td><p>13: if142014-09-04.013.fcs</p></td><td><p>G-1</p></td><td><p>- aTc</p></td><td><p>15.7</p></td><td><p></p></td></tr><tr><td><p>14: if142014-09-04.014.fcs</p></td><td><p>G-2</p></td><td><p>- aTc</p></td><td><p>20.4</p></td><td><p></p></td></tr><tr><td><p>15: if142014-09-04.015.fcs</p></td><td><p>G-3</p></td><td><p>- aTc</p></td><td><p>16.8</p></td><td><p></p></td></tr><tr><td><p>16: if142014-09-04.016.fcs</p></td><td><p>G-4</p></td><td><p>- aTc</p></td><td><p>17.1</p></td><td><p></p></td></tr><tr><td><p>17: if142014-09-04.017.fcs</p></td><td><p>C-1</p></td><td><p>- aTc</p></td><td><p>183</p></td><td><p></p></td></tr><tr><td><p>18: if142014-09-04.018.fcs</p></td><td><p>C-2</p></td><td><p>- aTc</p></td><td><p>182</p></td><td><p></p></td></tr><tr><td><p>19: if142014-09-04.019.fcs</p></td><td><p>C-3</p></td><td><p>- aTc</p></td><td><p>232</p></td><td><p></p></td></tr><tr><td><p>20: if142014-09-04.020.fcs</p></td><td><p>C-4</p></td><td><p>- aTc</p></td><td><p>186</p></td><td><p></p></td></tr></tbody></table><ul><li>aTc treatment seems to be moving fluor in the opposite direction of what we&rsquo;d expect of antitracr was doing its job. The degree is inconsistent, reflow is called for. </li></ul><p></p><p></p><p></p><p></p><hr style="page-break-before:always;display:none;"><p></p><hr style="page-break-before:always;display:none;"><p></p><p></p><h2><a name="h.ohhk3mdbkmcm"></a>9/8/14</h2><p>Objective: retest backbone shuffled antitracr bacteria</p><ul><li>Yesterday picked 4 new colonies from plates and 4 from 9/4/14 frozen stocks and grew in LB + antibiotics overnight. Diluted in to +/- aTc this morning and let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS</li><li>G1-4 are old frozen coli with K608012, pdCas9-GFP1, and 1A2-R0040-antitracr, C1-4 are old frozen with pdCas9. clones 5-8 are new from plates. AC are no-fluor (1A2 and pdCas9) but they were diluted in to triple antibiotic medium instead of double so they did not grow and are not reliable indicators of bg fluorescence on this run.</li><li>After flow, isolated singlets with flowjo (smalls) or left alone (bugs) and got gfpa unmodified or normalized by fsca or ssca. </li><li></li></ul><a href="#" name="777f62c089f7173b14ec6d88236db6c3e53afff8"></a><a href="#" name="4"></a><table cellpadding="0" cellspacing="0"<><tbody><tr><td><p></p></td><td><p></p></td><td><p></p></td><td><p></p></td><td><p>smalls</p></td><td><p></p></td><td><p></p></td><td><p>bugs</p></td><td><p></p></td></tr><tr><td><p></p></td><td><p></p></td><td><p>aTc</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td></tr><tr><td><p>1: if142014-09-08.001.fcs</p></td><td><p>G1</p></td><td><p>+</p></td><td><p>5.25</p></td><td><p>243</p></td><td><p>112</p></td><td><p>7.79</p></td><td><p>247</p></td><td><p>114</p></td></tr><tr><td><p>2: if142014-09-08.002.fcs</p></td><td><p>G2</p></td><td><p>+</p></td><td><p>4.41</p></td><td><p>226</p></td><td><p>78.1</p></td><td><p>7.12</p></td><td><p>228</p></td><td><p>76.2</p></td></tr><tr><td><p>3: if142014-09-08.003.fcs</p></td><td><p>G3</p></td><td><p>+</p></td><td><p>4.66</p></td><td><p>231</p></td><td><p>84.8</p></td><td><p>7.8</p></td><td><p>231</p></td><td><p>83.4</p></td></tr><tr><td><p>4: if142014-09-08.004.fcs</p></td><td><p>G4</p></td><td><p>+</p></td><td><p>3.86</p></td><td><p>191</p></td><td><p>70.1</p></td><td><p>5.99</p></td><td><p>192</p></td><td><p>67.9</p></td></tr><tr><td><p>5: if142014-09-08.005.fcs</p></td><td><p>G1</p></td><td><p>-</p></td><td><p>6.81</p></td><td><p>311</p></td><td><p>145</p></td><td><p>8.56</p></td><td><p>310</p></td><td><p>143</p></td></tr><tr><td><p>6: if142014-09-08.006.fcs</p></td><td><p>G2</p></td><td><p>-</p></td><td><p>6.37</p></td><td><p>337</p></td><td><p>153</p></td><td><p>7.25</p></td><td><p>323</p></td><td><p>145</p></td></tr><tr><td><p>7: if142014-09-08.007.fcs</p></td><td><p>G3</p></td><td><p>-</p></td><td><p>6.5</p></td><td><p>318</p></td><td><p>143</p></td><td><p>8.24</p></td><td><p>305</p></td><td><p>136</p></td></tr><tr><td><p>8: if142014-09-08.008.fcs</p></td><td><p>G4</p></td><td><p>-</p></td><td><p>6.14</p></td><td><p>298</p></td><td><p>140</p></td><td><p>7.66</p></td><td><p>288</p></td><td><p>131</p></td></tr><tr><td><p>9: if142014-09-08.009.fcs</p></td><td><p>C1</p></td><td><p>+</p></td><td><p>88.9</p></td><td><p>4071</p></td><td><p>1777</p></td><td><p>117</p></td><td><p>3978</p></td><td><p>1706</p></td></tr><tr><td><p>10: if142014-09-08.010.fcs</p></td><td><p>C2</p></td><td><p>+</p></td><td><p>83.5</p></td><td><p>4073</p></td><td><p>1547</p></td><td><p>120</p></td><td><p>3969</p></td><td><p>1422</p></td></tr><tr><td><p>11: if142014-09-08.011.fcs</p></td><td><p>C3</p></td><td><p>+</p></td><td><p>81.7</p></td><td><p>3957</p></td><td><p>1551</p></td><td><p>115</p></td><td><p>3840</p></td><td><p>1437</p></td></tr><tr><td><p>12: if142014-09-08.012.fcs</p></td><td><p>C4</p></td><td><p>+</p></td><td><p>77.2</p></td><td><p>3915</p></td><td><p>1577</p></td><td><p>95.7</p></td><td><p>3883</p></td><td><p>1503</p></td></tr><tr><td><p>13: if142014-09-08.013.fcs</p></td><td><p>C1</p></td><td><p>-</p></td><td><p>77.7</p></td><td><p>3686</p></td><td><p>1471</p></td><td><p>100</p></td><td><p>3545</p></td><td><p>1411</p></td></tr><tr><td><p>14: if142014-09-08.014.fcs</p></td><td><p>C2</p></td><td><p>-</p></td><td><p>64.5</p></td><td><p>3093</p></td><td><p>1445</p></td><td><p>88.3</p></td><td><p>2962</p></td><td><p>1373</p></td></tr><tr><td><p>15: if142014-09-08.015.fcs</p></td><td><p>C3</p></td><td><p>-</p></td><td><p>80.9</p></td><td><p>3971</p></td><td><p>1541</p></td><td><p>106</p></td><td><p>3771</p></td><td><p>1432</p></td></tr><tr><td><p>16: if142014-09-08.016.fcs</p></td><td><p>C4</p></td><td><p>-</p></td><td><p>58.9</p></td><td><p>2899</p></td><td><p>1299</p></td><td><p>79.7</p></td><td><p>2727</p></td><td><p>1220</p></td></tr><tr><td><p>17: if142014-09-08.017.fcs</p></td><td><p>G5</p></td><td><p>+</p></td><td><p>4.1</p></td><td><p>210</p></td><td><p>79.1</p></td><td><p>5.4</p></td><td><p>209</p></td><td><p>76.2</p></td></tr><tr><td><p>18: if142014-09-08.018.fcs</p></td><td><p>G6</p></td><td><p>+</p></td><td><p>4.9</p></td><td><p>225</p></td><td><p>98.2</p></td><td><p>7.07</p></td><td><p>226</p></td><td><p>97.6</p></td></tr><tr><td><p>19: if142014-09-08.019.fcs</p></td><td><p>G7</p></td><td><p>+</p></td><td><p>4.42</p></td><td><p>220</p></td><td><p>84.7</p></td><td><p>5.97</p></td><td><p>218</p></td><td><p>81.7</p></td></tr><tr><td><p>20: if142014-09-08.020.fcs</p></td><td><p>G8</p></td><td><p>+</p></td><td><p>4.19</p></td><td><p>211</p></td><td><p>81.3</p></td><td><p>5.68</p></td><td><p>213</p></td><td><p>79.5</p></td></tr><tr><td><p>21: if142014-09-08.021.fcs</p></td><td><p>G5</p></td><td><p>-</p></td><td><p>9.73</p></td><td><p>477</p></td><td><p>201</p></td><td><p>12.5</p></td><td><p>455</p></td><td><p>190</p></td></tr><tr><td><p>22: if142014-09-08.022.fcs</p></td><td><p>G6</p></td><td><p>-</p></td><td><p>9.68</p></td><td><p>450</p></td><td><p>197</p></td><td><p>12.9</p></td><td><p>449</p></td><td><p>196</p></td></tr><tr><td><p>23: if142014-09-08.023.fcs</p></td><td><p>G7</p></td><td><p>-</p></td><td><p>10.1</p></td><td><p>482</p></td><td><p>212</p></td><td><p>13.3</p></td><td><p>464</p></td><td><p>198</p></td></tr><tr><td><p>24: if142014-09-08.024.fcs</p></td><td><p>G8</p></td><td><p>-</p></td><td><p>10.1</p></td><td><p>489</p></td><td><p>216</p></td><td><p>13.3</p></td><td><p>473</p></td><td><p>204</p></td></tr><tr><td><p>25: if142014-09-08.025.fcs</p></td><td><p>C5</p></td><td><p>+</p></td><td><p>79.6</p></td><td><p>3958</p></td><td><p>1486</p></td><td><p>113</p></td><td><p>3813</p></td><td><p>1374</p></td></tr><tr><td><p>26: if142014-09-08.026.fcs</p></td><td><p>C6</p></td><td><p>+</p></td><td><p>80.8</p></td><td><p>4045</p></td><td><p>1514</p></td><td><p>113</p></td><td><p>4043</p></td><td><p>1425</p></td></tr><tr><td><p>27: if142014-09-08.027.fcs</p></td><td><p>C7</p></td><td><p>+</p></td><td><p>80.7</p></td><td><p>4053</p></td><td><p>1491</p></td><td><p>116</p></td><td><p>3921</p></td><td><p>1375</p></td></tr><tr><td><p>28: if142014-09-08.028.fcs</p></td><td><p>C8</p></td><td><p>+</p></td><td><p>79.8</p></td><td><p>3804</p></td><td><p>1555</p></td><td><p>103</p></td><td><p>3634</p></td><td><p>1508</p></td></tr><tr><td><p>29: if142014-09-08.029.fcs</p></td><td><p>C5</p></td><td><p>-</p></td><td><p>68.9</p></td><td><p>3268</p></td><td><p>1505</p></td><td><p>95.7</p></td><td><p>3128</p></td><td><p>1419</p></td></tr><tr><td><p>30: if142014-09-08.030.fcs</p></td><td><p>C6</p></td><td><p>-</p></td><td><p>80.2</p></td><td><p>3806</p></td><td><p>1639</p></td><td><p>110</p></td><td><p>3630</p></td><td><p>1541</p></td></tr><tr><td><p>31: if142014-09-08.031.fcs</p></td><td><p>C7</p></td><td><p>-</p></td><td><p>67.8</p></td><td><p>3240</p></td><td><p>1498</p></td><td><p>91.7</p></td><td><p>3041</p></td><td><p>1412</p></td></tr><tr><td><p>32: if142014-09-08.032.fcs</p></td><td><p>C8</p></td><td><p>-</p></td><td><p>83.7</p></td><td><p>3810</p></td><td><p>1675</p></td><td><p>107</p></td><td><p>3618</p></td><td><p>1621</p></td></tr><tr><td><p>33: if142014-09-08.033.fcs</p></td><td><p>AC1</p></td><td><p>+</p></td><td><p>0.895</p></td><td><p>40.2</p></td><td><p>16.8</p></td><td><p>0.854</p></td><td><p>34.9</p></td><td><p>12.9</p></td></tr><tr><td><p>34: if142014-09-08.034.fcs</p></td><td><p>AC3</p></td><td><p>+</p></td><td><p>0.539</p></td><td><p>28.4</p></td><td><p>11</p></td><td><p>0.785</p></td><td><p>27.4</p></td><td><p>10.7</p></td></tr><tr><td><p>35: if142014-09-08.035.fcs</p></td><td><p>AC1</p></td><td><p>-</p></td><td><p>0.464</p></td><td><p>25.8</p></td><td><p>9.39</p></td><td><p>0.605</p></td><td><p>25.9</p></td><td><p>8.65</p></td></tr><tr><td><p>36: if142014-09-08.036.fcs</p></td><td><p>AC3</p></td><td><p>-</p></td><td><p>0.532</p></td><td><p>28.3</p></td><td><p>10.4</p></td><td><p>0.661</p></td><td><p>26.2</p></td><td><p>9.23</p></td></tr></tbody></table><p></p><ul><li>Got ratios of fluorescence in +/- aTc and am reporting them in the table below</li></ul><a href="#" name="803fb1152dc946dc3b98f720d1637f36481109e8"></a><a href="#" name="5"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>n/a</p></td><td><p>n/a</p></td><td><p>+/-</p></td><td><p>smalls</p></td><td><p></p></td><td><p></p></td><td><p>bugs</p></td><td><p></p></td></tr><tr><td><p>n/a</p></td><td><p>n/a</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td><td><p>gfpa</p></td><td><p>gfpa/fsca*1000</p></td><td><p>gfpa/ssca*1000</p></td></tr><tr><td><p>1: if142014-09-08.001.fcs</p></td><td><p>G1</p></td><td><p>0.771</p></td><td><p>0.781</p></td><td><p>0.772</p></td><td><p>0.910</p></td><td><p>0.797</p></td><td><p>0.797</p></td></tr><tr><td><p>2: if142014-09-08.002.fcs</p></td><td><p>G2</p></td><td><p>0.692</p></td><td><p>0.671</p></td><td><p>0.510</p></td><td><p>0.982</p></td><td><p>0.706</p></td><td><p>0.526</p></td></tr><tr><td><p>3: if142014-09-08.003.fcs</p></td><td><p>G3</p></td><td><p>0.717</p></td><td><p>0.726</p></td><td><p>0.593</p></td><td><p>0.947</p></td><td><p>0.757</p></td><td><p>0.613</p></td></tr><tr><td><p>4: if142014-09-08.004.fcs</p></td><td><p>G4</p></td><td><p>0.629</p></td><td><p>0.641</p></td><td><p>0.501</p></td><td><p>0.782</p></td><td><p>0.667</p></td><td><p>0.518</p></td></tr><tr><td><p>9: if142014-09-08.009.fcs</p></td><td><p>C1</p></td><td><p>1.144</p></td><td><p>1.104</p></td><td><p>1.208</p></td><td><p>1.170</p></td><td><p>1.122</p></td><td><p>1.209</p></td></tr><tr><td><p>10: if142014-09-08.010.fcs</p></td><td><p>C2</p></td><td><p>1.295</p></td><td><p>1.317</p></td><td><p>1.071</p></td><td><p>1.359</p></td><td><p>1.340</p></td><td><p>1.036</p></td></tr><tr><td><p>11: if142014-09-08.011.fcs</p></td><td><p>C3</p></td><td><p>1.010</p></td><td><p>0.996</p></td><td><p>1.006</p></td><td><p>1.085</p></td><td><p>1.018</p></td><td><p>1.003</p></td></tr><tr><td><p>12: if142014-09-08.012.fcs</p></td><td><p>C4</p></td><td><p>1.311</p></td><td><p>1.350</p></td><td><p>1.214</p></td><td><p>1.201</p></td><td><p>1.424</p></td><td><p>1.232</p></td></tr><tr><td><p>17: if142014-09-08.017.fcs</p></td><td><p>G5</p></td><td><p>0.421</p></td><td><p>0.440</p></td><td><p>0.394</p></td><td><p>0.432</p></td><td><p>0.459</p></td><td><p>0.401</p></td></tr><tr><td><p>18: if142014-09-08.018.fcs</p></td><td><p>G6</p></td><td><p>0.506</p></td><td><p>0.500</p></td><td><p>0.498</p></td><td><p>0.548</p></td><td><p>0.503</p></td><td><p>0.498</p></td></tr><tr><td><p>19: if142014-09-08.019.fcs</p></td><td><p>G7</p></td><td><p>0.438</p></td><td><p>0.456</p></td><td><p>0.400</p></td><td><p>0.449</p></td><td><p>0.470</p></td><td><p>0.413</p></td></tr><tr><td><p>20: if142014-09-08.020.fcs</p></td><td><p>G8</p></td><td><p>0.415</p></td><td><p>0.431</p></td><td><p>0.376</p></td><td><p>0.427</p></td><td><p>0.450</p></td><td><p>0.390</p></td></tr><tr><td><p>25: if142014-09-08.025.fcs</p></td><td><p>C5</p></td><td><p>1.155</p></td><td><p>1.211</p></td><td><p>0.987</p></td><td><p>1.181</p></td><td><p>1.219</p></td><td><p>0.968</p></td></tr><tr><td><p>26: if142014-09-08.026.fcs</p></td><td><p>C6</p></td><td><p>1.007</p></td><td><p>1.063</p></td><td><p>0.924</p></td><td><p>1.027</p></td><td><p>1.114</p></td><td><p>0.925</p></td></tr><tr><td><p>27: if142014-09-08.027.fcs</p></td><td><p>C7</p></td><td><p>1.190</p></td><td><p>1.251</p></td><td><p>0.995</p></td><td><p>1.265</p></td><td><p>1.289</p></td><td><p>0.974</p></td></tr><tr><td><p>28: if142014-09-08.028.fcs</p></td><td><p>C8</p></td><td><p>0.953</p></td><td><p>0.998</p></td><td><p>0.928</p></td><td><p>0.963</p></td><td><p>1.004</p></td><td><p>0.930</p></td></tr><tr><td><p>33: if142014-09-08.033.fcs</p></td><td><p>AC1</p></td><td><p>1.929</p></td><td><p>1.558</p></td><td><p>1.789</p></td><td><p>1.412</p></td><td><p>1.347</p></td><td><p>1.491</p></td></tr><tr><td><p>34: if142014-09-08.034.fcs</p></td><td><p>AC3</p></td><td><p>1.013</p></td><td><p>1.004</p></td><td><p>1.058</p></td><td><p>1.188</p></td><td><p>1.046</p></td><td><p>1.159</p></td></tr></tbody></table><ul><li></li></ul><hr style="page-break-before:always;display:none;"><p><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 560.00px; height: 420.00px;"><img alt="" src="https://2014.igem.org/File:9-4-14-2.jpg" style="width: 560.00px; height: 420.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></p><p>Box plot of just G1-4 and C1-4. Note that the G clones from plates (clones 5-8) have even smaller ratios. </p><hr style="page-break-before:always;display:none;"><p></p><p></p><p></p><p></p><hr style="page-break-before:always;display:none;"><p></p><p></p><p></p><p></p><h2><a name="h.h7pf932a50dt"></a>9/9/14</h2><p>Objective: assembly decoy binding site arrays</p><ul><li>Garima sets up a PCR and is asked to fill in notes about it.</li><li>Results</li><li>Charlie sets up a new set of tubes for PCR with a 5 rx master mix containing</li></ul><ul><li>190uL H2O</li><li>50 uL Q5 buffer</li><li>2.0 uL oligo 1 (gfp1-spacer-gfp1)</li><li>2.5 uL Q5 poly</li><li>5 uL dNTPs</li></ul><ul><li>And then divides that in to four tubes before adding in the following volumes of oligo 2 (spacer-gfp1-space)</li></ul><a href="#" name="fd71260aa00de3f043dfc426450b7d96e13e45b0"></a><a href="#" name="6"></a><table cellpadding="0" cellspacing="0"><tbody><tr><td><p>tube number</p></td><td><p>1</p></td><td><p>2</p></td><td><p>3</p></td><td><p>4</p></td></tr><tr><td><p>uL oligo 2</p></td><td><p>0</p></td><td><p>0.5</p></td><td><p>1.0</p></td><td><p>2.0</p></td></tr></tbody></table><ul><li><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 624.00px; height: 468.00px;"><img alt="Sep 10, 2014 1:24:31 PM.jpg" src="https://static.igem.org/mediawiki/2014/a/a4/9-4-14-3.jpg" style="width: 624.00px; height: 468.00px; margin-left: 0.00px; margin-top: 0.00px; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px);" title=""></li></ul><ul><li>Streakiness on the row with 0.5uL of oligo 2 looks promising? IGEM protocol on the thermal cycler modified</li></ul><h2><a name="h.3838tuwxw3ds"></a>
</html>
</html>

Revision as of 04:16, 17 October 2014

9/4/14

Objective: test backbone shuffled antitracr bacteria

  • Started colonies from plates and diluted in to +/- aTc this morning. Let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS for flow.
  • Took data to flowjo and plotted histograms below. This is ungated fluorescence
  • table with mean GFPAs

ungated GFPA

+/- aTc

1: if142014-09-04.001.fcs

AC-1

+ aTc

0.402

1.185840708

2: if142014-09-04.002.fcs

AC-2

+ aTc

0.455

1.197368421

3: if142014-09-04.003.fcs

G-1

+ aTc

14.9

0.949044586

4: if142014-09-04.004.fcs

G-2

+ aTc

11.7

0.573529412

5: if142014-09-04.005.fcs

G-3

+ aTc

8.91

0.530357143

6: if142014-09-04.006.fcs

G-4

+ aTc

5.92

0.34619883

7: if142014-09-04.007.fcs

C-1

+ aTc

186

1.016393443

8: if142014-09-04.008.fcs

C-2

+ aTc

224

1.230769231

9: if142014-09-04.009.fcs

C-3

+ aTc

214

0.922413793

10: if142014-09-04.010.fcs

C-4

+ aTc

222

1.193548387

11: if142014-09-04.011.fcs

AC-1

- aTc

0.339

12: if142014-09-04.012.fcs

AC-2

- aTc

0.38

13: if142014-09-04.013.fcs

G-1

- aTc

15.7

14: if142014-09-04.014.fcs

G-2

- aTc

20.4

15: if142014-09-04.015.fcs

G-3

- aTc

16.8

16: if142014-09-04.016.fcs

G-4

- aTc

17.1

17: if142014-09-04.017.fcs

C-1

- aTc

183

18: if142014-09-04.018.fcs

C-2

- aTc

182

19: if142014-09-04.019.fcs

C-3

- aTc

232

20: if142014-09-04.020.fcs

C-4

- aTc

186

  • aTc treatment seems to be moving fluor in the opposite direction of what we’d expect of antitracr was doing its job. The degree is inconsistent, reflow is called for.



9/8/14

Objective: retest backbone shuffled antitracr bacteria

  • Yesterday picked 4 new colonies from plates and 4 from 9/4/14 frozen stocks and grew in LB + antibiotics overnight. Diluted in to +/- aTc this morning and let shake for 5(?) hours before collecting 500uL and spin/resusp in PBS
  • G1-4 are old frozen coli with K608012, pdCas9-GFP1, and 1A2-R0040-antitracr, C1-4 are old frozen with pdCas9. clones 5-8 are new from plates. AC are no-fluor (1A2 and pdCas9) but they were diluted in to triple antibiotic medium instead of double so they did not grow and are not reliable indicators of bg fluorescence on this run.
  • After flow, isolated singlets with flowjo (smalls) or left alone (bugs) and got gfpa unmodified or normalized by fsca or ssca.

smalls

bugs

aTc

gfpa

gfpa/fsca*1000

gfpa/ssca*1000

gfpa

gfpa/fsca*1000

gfpa/ssca*1000

1: if142014-09-08.001.fcs

G1

+

5.25

243

112

7.79

247

114

2: if142014-09-08.002.fcs

G2

+

4.41

226

78.1

7.12

228

76.2

3: if142014-09-08.003.fcs

G3

+

4.66

231

84.8

7.8

231

83.4

4: if142014-09-08.004.fcs

G4

+

3.86

191

70.1

5.99

192

67.9

5: if142014-09-08.005.fcs

G1

-

6.81

311

145

8.56

310

143

6: if142014-09-08.006.fcs

G2

-

6.37

337

153

7.25

323

145

7: if142014-09-08.007.fcs

G3

-

6.5

318

143

8.24

305

136

8: if142014-09-08.008.fcs

G4

-

6.14

298

140

7.66

288

131

9: if142014-09-08.009.fcs

C1

+

88.9

4071

1777

117

3978

1706

10: if142014-09-08.010.fcs

C2

+

83.5

4073

1547

120

3969

1422

11: if142014-09-08.011.fcs

C3

+

81.7

3957

1551

115

3840

1437

12: if142014-09-08.012.fcs

C4

+

77.2

3915

1577

95.7

3883

1503

13: if142014-09-08.013.fcs

C1

-

77.7

3686

1471

100

3545

1411

14: if142014-09-08.014.fcs

C2

-

64.5

3093

1445

88.3

2962

1373

15: if142014-09-08.015.fcs

C3

-

80.9

3971

1541

106

3771

1432

16: if142014-09-08.016.fcs

C4

-

58.9

2899

1299

79.7

2727

1220

17: if142014-09-08.017.fcs

G5

+

4.1

210

79.1

5.4

209

76.2

18: if142014-09-08.018.fcs

G6

+

4.9

225

98.2

7.07

226

97.6

19: if142014-09-08.019.fcs

G7

+

4.42

220

84.7

5.97

218

81.7

20: if142014-09-08.020.fcs

G8

+

4.19

211

81.3

5.68

213

79.5

21: if142014-09-08.021.fcs

G5

-

9.73

477

201

12.5

455

190

22: if142014-09-08.022.fcs

G6

-

9.68

450

197

12.9

449

196

23: if142014-09-08.023.fcs

G7

-

10.1

482

212

13.3

464

198

24: if142014-09-08.024.fcs

G8

-

10.1

489

216

13.3

473

204

25: if142014-09-08.025.fcs

C5

+

79.6

3958

1486

113

3813

1374

26: if142014-09-08.026.fcs

C6

+

80.8

4045

1514

113

4043

1425

27: if142014-09-08.027.fcs

C7

+

80.7

4053

1491

116

3921

1375

28: if142014-09-08.028.fcs

C8

+

79.8

3804

1555

103

3634

1508

29: if142014-09-08.029.fcs

C5

-

68.9

3268

1505

95.7

3128

1419

30: if142014-09-08.030.fcs

C6

-

80.2

3806

1639

110

3630

1541

31: if142014-09-08.031.fcs

C7

-

67.8

3240

1498

91.7

3041

1412

32: if142014-09-08.032.fcs

C8

-

83.7

3810

1675

107

3618

1621

33: if142014-09-08.033.fcs

AC1

+

0.895

40.2

16.8

0.854

34.9

12.9

34: if142014-09-08.034.fcs

AC3

+

0.539

28.4

11

0.785

27.4

10.7

35: if142014-09-08.035.fcs

AC1

-

0.464

25.8

9.39

0.605

25.9

8.65

36: if142014-09-08.036.fcs

AC3

-

0.532

28.3

10.4

0.661

26.2

9.23

  • Got ratios of fluorescence in +/- aTc and am reporting them in the table below

n/a

n/a

+/-

smalls

bugs

n/a

n/a

gfpa

gfpa/fsca*1000

gfpa/ssca*1000

gfpa

gfpa/fsca*1000

gfpa/ssca*1000

1: if142014-09-08.001.fcs

G1

0.771

0.781

0.772

0.910

0.797

0.797

2: if142014-09-08.002.fcs

G2

0.692

0.671

0.510

0.982

0.706

0.526

3: if142014-09-08.003.fcs

G3

0.717

0.726

0.593

0.947

0.757

0.613

4: if142014-09-08.004.fcs

G4

0.629

0.641

0.501

0.782

0.667

0.518

9: if142014-09-08.009.fcs

C1

1.144

1.104

1.208

1.170

1.122

1.209

10: if142014-09-08.010.fcs

C2

1.295

1.317

1.071

1.359

1.340

1.036

11: if142014-09-08.011.fcs

C3

1.010

0.996

1.006

1.085

1.018

1.003

12: if142014-09-08.012.fcs

C4

1.311

1.350

1.214

1.201

1.424

1.232

17: if142014-09-08.017.fcs

G5

0.421

0.440

0.394

0.432

0.459

0.401

18: if142014-09-08.018.fcs

G6

0.506

0.500

0.498

0.548

0.503

0.498

19: if142014-09-08.019.fcs

G7

0.438

0.456

0.400

0.449

0.470

0.413

20: if142014-09-08.020.fcs

G8

0.415

0.431

0.376

0.427

0.450

0.390

25: if142014-09-08.025.fcs

C5

1.155

1.211

0.987

1.181

1.219

0.968

26: if142014-09-08.026.fcs

C6

1.007

1.063

0.924

1.027

1.114

0.925

27: if142014-09-08.027.fcs

C7

1.190

1.251

0.995

1.265

1.289

0.974

28: if142014-09-08.028.fcs

C8

0.953

0.998

0.928

0.963

1.004

0.930

33: if142014-09-08.033.fcs

AC1

1.929

1.558

1.789

1.412

1.347

1.491

34: if142014-09-08.034.fcs

AC3

1.013

1.004

1.058

1.188

1.046

1.159


Box plot of just G1-4 and C1-4. Note that the G clones from plates (clones 5-8) have even smaller ratios.



9/9/14

Objective: assembly decoy binding site arrays

  • Garima sets up a PCR and is asked to fill in notes about it.
  • Results
  • Charlie sets up a new set of tubes for PCR with a 5 rx master mix containing
  • 190uL H2O
  • 50 uL Q5 buffer
  • 2.0 uL oligo 1 (gfp1-spacer-gfp1)
  • 2.5 uL Q5 poly
  • 5 uL dNTPs
  • And then divides that in to four tubes before adding in the following volumes of oligo 2 (spacer-gfp1-space)

tube number

1

2

3

4

uL oligo 2

0

0.5

1.0

2.0

  • Sep 10, 2014 1:24:31 PM.jpg
  • Streakiness on the row with 0.5uL of oligo 2 looks promising? IGEM protocol on the thermal cycler modified