Team:IPN-UNAM-Mexico/Notebook/August 11 2009

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                                  <td><p>&nbsp;</p>
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                                    <p><strong>August 11th, 2009</strong></p>
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                                    <p>Amplification of the promoter and RBS using  plasmidic DNA from Q04121.</p>
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                                    <blockquote>
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                                      <p>Digestion Mix<br>
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PlasmidicDNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;  4.0 &mu;l (= 1 &mu;g  DNA)&nbsp; x 20 = 80 &mu;l<br>
 +
BSA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;  0.5 &mu;l&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; x 20 = 10 &mu;l<br>
 +
Buffer 2 &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;1.0 &mu;l&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; x 20 = 20 &mu;l<br>
 +
Enzyme EcoRI &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;0.5  &mu;l&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;x 20 = 10 &mu;l<br>
 +
H2O &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;4.0 &mu;l&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; x 20 = 80 &mu;l&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <br>
 +
Cf&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10.0 &mu;l &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 200 &mu;l</p>
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                                    </blockquote>
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                                    <p>&nbsp;</p>
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                                    <p>Load order:</p>
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                                    <blockquote>
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                                      <p>Well&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Plasmid<br>
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                                        2&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2:3  L1<br>
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                                        3&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2:3  L2&rsquo;<br>
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                                        4&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2:3  L2&rdquo;<br>
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                                        5&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 15:13<br>
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                                        6&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 15:3<br>
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                                        7&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4:23<br>
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                                        8&nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4:23  L2</p>
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                                    </blockquote>
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                                    <p>&nbsp;</p>
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                                    <blockquote>
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                                      <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<strong>Gel 7:31 pm</strong><br>
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                                        Well&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Plasmid&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Well&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Plasmid<br>
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                                        1&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Marker&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; Marker<br>
 +
                                        2 &nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2_3L2#1&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp; 2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp; 9.1<br>
 +
                                        3&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2:3L2#2&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.3<br>
 +
                                        4&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 2_3L2#3&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp; 4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.4<br>
 +
                                        5&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 3.3*&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.5<br>
 +
                                        6&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4_23L1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 6&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.6<br>
 +
                                        7&nbsp; No&nbsp;&nbsp;&nbsp; 4_23L2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 7&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.7<br>
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                                        8&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 7.3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 8&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.8<br>
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                                        9&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 11.1&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.9<br>
 +
                                        10&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 14.2&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 9.10<br>
 +
                                        11&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 15.3&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 11&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Marker<br>
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                                        12&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 15_13L2  #1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 12&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; ----------<br>
 +
                                        13&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 15_13L2  #2&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Digest with EcoRI<br>
 +
                                        14&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 15_13L2  #3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; at 4&deg;C<br>
 +
                                        15&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 16.2&rdquo;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Digest with EcoRI<br>
 +
                                        16&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 18.2*<br>
 +
                                        17&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 19.1*&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Repeat  digestion &ndash;DNA<br>
 +
                                        18&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 22.1*&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Repeat  digestion-DNA<br>
 +
                                        19&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 24.2&rdquo;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Repeat  digestion &ndash; DNA<br>
 +
                                        20&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Marker</p>
 +
                                      <p>&nbsp;Note: it is needed to digest  lesser the plasmid 11.1.</p>
 +
                                    </blockquote>
 +
                                    <p>&nbsp;</p>
 +
                                    <p>Repeat  digestions of the plasmids:</p>
 +
                                    <ul>
 +
                                      <li>19.1 </li>
 +
                                      <li>22.1</li>
 +
                                      <li>24.2</li>
 +
                                    </ul>
 +
                                    <p>which are  at&nbsp; -20&deg;C in the Unicel (with 3&mu;l of  DNA) and the 9 is at 4&deg;C.<br>
 +
                                      Put 3 &mu;l of DNA  and 5 &mu;l of water.<br>
 +
                                      Digestion Mix x  10.</p>
 +
                                    <p align="left" class="style8">&nbsp;</p>
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                                    <p>&nbsp;</p>
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                                    <p align="left" >&nbsp;</p>
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                                    <p align="left">&nbsp;</p>
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August 11th, 2009

Amplification of the promoter and RBS using plasmidic DNA from Q04121.

Digestion Mix
PlasmidicDNA             4.0 μl (= 1 μg DNA)  x 20 = 80 μl
BSA                          0.5 μl                     x 20 = 10 μl
Buffer 2                    1.0 μl                      x 20 = 20 μl
Enzyme EcoRI            0.5 μl                      x 20 = 10 μl
H2O                         4.0 μl                       x 20 = 80 μl                
Cf                            10.0 μl                          200 μl

 

Load order:

Well                 Plasmid
2                      2:3 L1
3                      2:3 L2’
4                      2:3 L2”
5                      15:13
6                      15:3
7                      4:23
8                      4:23 L2

 

                        Gel 7:31 pm
Well      Plasmid            Well           Plasmid
1          Marker              1              Marker
2          2_3L2#1           2               9.1
3          2:3L2#2            3                9.3
4          2_3L2#3           4                9.4
5          3.3*                 5                9.5
6          4_23L1             6                9.6
7  No    4_23L2             7                9.7
8          7.3                   8                9.8
9          11.1                 9                9.9
10        14.2                 10              9.10
11        15.3                 11             Marker
12        15_13L2 #1      12              ----------
13        15_13L2 #2         Digest with EcoRI
14        15_13L2 #3               at 4°C
15        16.2”                  Digest with EcoRI
16        18.2*
17        19.1*                Repeat digestion –DNA
18        22.1*                Repeat digestion-DNA
19        24.2”                Repeat digestion – DNA
20        Marker

 Note: it is needed to digest lesser the plasmid 11.1.

 

Repeat digestions of the plasmids:

  • 19.1
  • 22.1
  • 24.2

which are at  -20°C in the Unicel (with 3μl of DNA) and the 9 is at 4°C.
Put 3 μl of DNA and 5 μl of water.
Digestion Mix x 10.

 

 

 

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