Team:Gothenburg/Calendar

From 2014.igem.org

(Difference between revisions)
(Styled the table a bit)
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#calendar>table {
#calendar>table {
margin: 2em;
margin: 2em;
-
border-spacing: 5px;
+
border-spacing: 10px;
border-collapse: separate;
border-collapse: separate;
}
}
-
+
 
-
#calendar table tr td {
+
-
width: 130px;
+
-
height: 80px;
+
-
}
+
-
+
.date {
.date {
background-color: #f4f4f4;
background-color: #f4f4f4;
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             margin-left: 1em;
             margin-left: 1em;
         }
         }
-
+
 
-
.prev-month, .next-month {
+
-
background-color: #7d7d7d;
+
-
}
+
-
+
         #contentDialog {
         #contentDialog {
             min-height: 100px;
             min-height: 100px;
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             height: 75%;
             height: 75%;
             padding: 16px;
             padding: 16px;
-
             border: 16px solid #ed6d22;
+
             border: 16px solid #ED6D22;
             background-color: white;
             background-color: white;
             z-index:1002;
             z-index:1002;
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                     <h1>26</h1>
                     <h1>26</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid Purification</li>
-
                         <li>Title two</li>
+
                         <li>Amplification and purification of parts</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid Purification</h2>
 +
<p> Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416, p2055 and a plasmid containig our Cas9 sequence
 +
(pTPG1-dCas9-UPGP)via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by
 +
water on the last step. <br>
 +
<h2>Amplification and purification of parts</h2>
 +
<p>Performed a PCR amplification of the non-synthetic parts that failed on the day before and on the dCas9 sequence and the three fluorescent proteins
 +
-Yellow, Cyano and Red (YFP, CFP and RFP, respectively). <br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC </li>
 +
<li>Gradient from 50 to 55ºC for 30s </li>
 +
<li>30s at 60ºC </li>
 +
<li>2min 15s at 72ºC </li>
 +
<li>Go to step 2 (repeat 39 times) </li>
 +
<li>10 min at 72ºC. </li>
 +
</ol>
 +
<p>A diagnostic gel was performed and the result is as follows: <br>
 +
[[File: Bio-Rad 2014-06-26 17hr 53min.jpg|frameless|300px]] <br>
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>27</h1>
                     <h1>27</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Gel extraction</li>
-
                        <li>Title two</li>
+
<li>PCR amplification and merging</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the fluorecent proteins amplified on the day before with a GeneJet gel extraction kit.The manufacter's
 +
instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>PCR amplification and merging</h2>
 +
<p>Performed a PCR to fuse the pDSE4, pHO and dCas9 parts together.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>Gradient from 50 to 55ºC for 30s</li>
 +
<li>30s at 60ºC</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 39 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p>A diagnostic gel was performed and the result is as follows: <br>
 +
[[File: Bio-Rad 2014-06-27 15hr 07min-2.jpg|frameless|300px]] <br>
 +
<p>The bands did not seem correct, so this PCR was repeated on the next day.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>28</h1>
                     <h1>28</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification and merging</li>
-
                         <li>Title two</li>
+
                         <li>Plasmid restriction</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
<p>Repeated the PCR to fuse the pDSE4, pHO and dCas9 parts together.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 39 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p>A diagnostic gel was performed and the result is as follows: <br>
 +
[File: Bio-Rad 2014-06-30 15hr 24min.jpg|frameless|300px]] <br>
 +
<p>This did not seemed to work either.
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>29</h1>
                     <h1>29</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
 
-
                        <li>Title two</li>
 
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
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                     <h1>30</h1>
                     <h1>30</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification and merging</li>
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
Repeated the PCR to fuse the pDSE4, pHO and dCas9 parts together.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 30s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p> A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p>Samples were loaded into a gelGreen, that showed p413, p415 and p416 approximately in the 5000bp size band, while p414 presented two bands, one around 4000bp and another at 2000bp.<br>
 +
<p> this indicates that the 5000bp band corresponds to the uncut plasmid, while the 4000bp and 2000bp are probably contaminations.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
-
                 <td id="2014-07-01" class="date next-month"> <!-- My birthday -->
+
                 <td id="2014-07-01" class="date next-month">  
                     <h1>1</h1>
                     <h1>1</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                      <li>Plasmid restriction</li>
-
                         <li>Title two</li>
+
                         <li>PCR amplification</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<h2>PCR amplification</h2>
 +
<p>Amplified the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
<p> A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>2</h1>
                     <h1>2</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification</li>
-
                         <li>Title two</li>
+
<li>Gel extraction</li>
 +
                         <li>Concentration determination</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification</h2>
 +
<p>Repeated the amplification of the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
<p> A diagnostic gel was performed and the sizes of the fragments matched for the pDSE4 and pHO, but not for the Cas9.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the DNA fragments previously amplified with a GeneJet gel extraction kit.The manufacter's
 +
instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of all the fragments obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 8.5 ng of DNA per UL to 42ng/UL.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>3</h1>
                     <h1>3</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                    </ul>
-
                        <li>Title two</li>
+
                     <span class="hidden"></span>
-
                    </ul>
+
-
                     <span class="hidden">This is where all the information goes!</span>
+
                 </td>
                 </td>
                  
                  
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                     <h1>4</h1>
                     <h1>4</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid Purification</li>
-
                         <li>Title two</li>
+
                         <li>Plasmid restriction</li>
 +
<li>Gel extraction</li>
 +
<li>Concentration determination</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid purification</h2>
 +
<p>Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416 and p2055 via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by
 +
water on the last step. Each plasmid was purified in duplicates.
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p> A diagnostic gelGreen was performed and showed that the cleavage of plasmids p413, p415 and p416 was successful.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of the circular and linear plasmids obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 6.55 ng of DNA per UL to 543.4ng/UL.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>5</h1>
                     <h1>5</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
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                     <h1>6</h1>
                     <h1>6</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
 
-
                        <li>Title two</li>
 
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>

Revision as of 21:39, 5 October 2014

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June

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  • Safety Instructions

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  • Introduction to centrifuges and autoclaves

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  • Finished mandatory training

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  • Media Preparation

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  • Amplification and Purification of non-synthetic parts

26

  • Plasmid Purification
  • Amplification and purification of parts

27

  • Gel extraction
  • PCR amplification and merging

28

  • PCR amplification and merging
  • Plasmid restriction

29

30

  • PCR amplification and merging

1

  • Plasmid restriction
  • PCR amplification

2

  • PCR amplification
  • Gel extraction
  • Concentration determination

3

4

  • Plasmid Purification
  • Plasmid restriction
  • Gel extraction
  • Concentration determination

5

6

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5

  • Title one
  • Title two

6

  • Title one
  • Title two

7

  • Title one
  • Title two

8

  • Title one
  • Title two

9

  • Title one
  • Title two

10

  • Title one
  • Title two

11

  • Title one
  • Title two

12

  • Title one
  • Title two

13

  • Title one
  • Title two

14

  • Title one
  • Title two

15

  • Title one
  • Title two

16

  • Title one
  • Title two

17

  • Title one
  • Title two

18

  • Title one
  • Title two

19

  • Title one
  • Title two

20

  • Title one
  • Title two

21

  • Title one
  • Title two

22

  • Title one
  • Title two

23

  • Title one
  • Title two

24

  • Title one
  • Title two

25

  • Title one
  • Title two

26

  • Title one
  • Title two

27

  • Title one
  • Title two

28

  • Title one
  • Title two

29

  • Title one
  • Title two

30

  • Title one
  • Title two

31

  • Title one
  • Title two

September

1

  • Title one
  • Title two

2

  • Title one
  • Title two

3

  • Title one
  • Title two

4

  • Title one
  • Title two

5

  • Title one
  • Title two

6

  • Title one
  • Title two

7

  • Title one
  • Title two

8

  • Title one
  • Title two

9

  • Title one
  • Title two

10

  • Title one
  • Title two

11

  • Title one
  • Title two

12

  • Title one
  • Title two

13

  • Title one
  • Title two

14

  • Title one
  • Title two

15

  • Title one
  • Title two

16

  • Title one
  • Title two

17

  • Title one
  • Title two

18

  • Title one
  • Title two

19

  • Title one
  • Title two

20

  • Title one
  • Title two

21

  • Title one
  • Title two

22

  • Title one
  • Title two

23

  • Title one
  • Title two

24

  • Title one
  • Title two

25

  • Title one
  • Title two

26

  • Title one
  • Title two

27

  • Title one
  • Title two

28

  • Title one
  • Title two

29

  • Title one
  • Title two

30

  • Title one
  • Title two

1

  • Title one
  • Title two

2

  • Title one
  • Title two

3

  • Title one
  • Title two

4

  • Title one
  • Title two

5

  • Title one
  • Title two

Retrieved from "http://2014.igem.org/Team:Gothenburg/Calendar"