Team:UNIPV-Pavia/Calendar/July/settimana3

From 2011.igem.org

(Difference between revisions)
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<p>
<p>
  As shown, in figure all clones were positive, so we cut and purified the bands of interest.
  As shown, in figure all clones were positive, so we cut and purified the bands of interest.
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</br>
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</p>
 +
<p>
After gel extraction, cut DNA was quantified:
After gel extraction, cut DNA was quantified:
</p>
</p>
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 +
 +
<center>
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<table border="1">
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    <tr>
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      <td><b>Plasmid</b></td>
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      <td><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 +
  <tr>
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      <td></html>E1<html> (X-P)</td>
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      <td>4.2</td>
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  </tr>
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 +
  <tr>
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      <td></html>E2<html> (X-P)</td>
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      <td>3.8</td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td></html>E3<html> (X-P)</td>
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      <td>4.1</td>
 +
  </tr>
 +
 +
  <tr>
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      <td></html>E4<html> (X-P)</td>
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      <td>4.2</td>
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  </tr>
 +
 +
 +
  <tr>
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      <td></html>E5<html> (X-P)</td>
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      <td>5.6</td>
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  </tr>
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 +
  <tr>
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      <td></html>E6<html> (X-P)</td>
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      <td>7.8</td>
 +
  </tr>
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 +
  <tr>
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      <td></html>E7<html> (X-P)</td>
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      <td>6.4</td>
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  </tr>
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 +
  <tr>
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      <td></html>E8<html> (S-P)</td>
 +
      <td>3.4</td>
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  </tr>
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 +
  <tr>
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      <td></html><partinfo>BBa_C0261</partinfo><html> (X-P)</td>
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      <td>4.8</td>
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  </tr>
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 +
  <tr>
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      <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
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      <td>7.9</td>
 +
  </tr>
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 +
  <tr>
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      <td></html><partinfo>BBa_B0034</partinfo><html> (S-P)</td>
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      <td>11.0</td>
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  </tr>
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 +
  <tr>
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      <td></html><partinfo>BBa_I13507</partinfo><html> (X-P)</td>
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      <td>3.9</td>
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  </tr>
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</table>
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</center>
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 +
<p>
 +
Then ligations were performed in a final volume of 10 &mu;l:
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</p>
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<center>
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<table border="1">
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    <tr>
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      <td><b>Ligation Name</b></td>
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      <td><b>Vector</b></td>
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      <td><b>Vector volume (&mu;l)</b></td>
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      <td><b>Insert</b></td>
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      <td><b>Insert volume (&mu;l)</b></td>
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      <td><b>Buffer (&mu;l)</b></td>
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      <td><b>T4 Ligase (&mu;l)</b></td>
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  </tr>
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    <tr>
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      <td><b>E9</b></td>
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      <td></html><partinfo>BBa_B0030</partinfo><html> (S-P)</td>
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      <td>1</td>
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      <td>E1 (X-P)</td>
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      <td>7</td>
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      <td>1</td>
 +
      <td>1</td>
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  </tr>
 +
 +
 +
    <tr>
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      <td><b>E10</b></td>
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      <td></html><partinfo>BBa_B0031</partinfo><html> (S-P)</td>
 +
      <td>1</td>
 +
      <td>E1 (X-P)</td>
 +
      <td>7</td>
 +
      <td>1</td>
 +
      <td>1</td>
 +
  </tr>
 +
 +
    <tr>
 +
      <td><b>E11</b></td>
 +
      <td></html><partinfo>BBa_B0032</partinfo><html> (S-P)</td>
 +
      <td>1</td>
 +
      <td>E1 (X-P)</td>
 +
      <td>7</td>
 +
      <td>1</td>
 +
      <td>1</td>
 +
  </tr>
 +
 +
    <tr>
 +
      <td><b>E12</b></td>
 +
      <td></html><partinfo>BBa_B0034</partinfo><html> (S-P)</td>
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      <td>1</td>
 +
      <td>E1 (X-P)</td>
 +
      <td>7</td>
 +
      <td>1</td>
 +
      <td>1</td>
 +
  </tr>
 +
 +
    <tr>
 +
      <td><b>E13</b></td>
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      <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
 +
      <td>1.5</td>
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      <td>E2 (X-P)</td>
 +
      <td>6.5</td>
 +
      <td>1</td>
 +
      <td>1</td>
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  </tr>
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 +
    <tr>
 +
      <td><b>E14</b></td>
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      <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
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      <td>1.5</td>
 +
      <td>E3 (X-P)</td>
 +
      <td>6.5</td>
 +
      <td>1</td>
 +
      <td>1</td>
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  </tr>
 +
 +
    <tr>
 +
      <td><b>E15</b></td>
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      <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
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      <td>1.5</td>
 +
      <td>E4 (X-P)</td>
 +
      <td>6.5</td>
 +
      <td>1</td>
 +
      <td>1</td>
 +
  </tr>
 +
 +
    <tr>
 +
      <td><b>E16</b></td>
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      <td></html><partinfo>BBa_R0040</partinfo><html> (S-P)</td>
 +
      <td>2</td>
 +
      <td></html><partinfo>BBa_C0261</partinfo><html> (X-P)</td>
 +
      <td>6</td>
 +
      <td>1</td>
 +
      <td>1</td>
 +
  </tr>
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 +
</table>
 +
</center>
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<br>
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<br>
<table border="0" width="100%" class="menu">
<table border="0" width="100%" class="menu">
<tr>
<tr>

Revision as of 22:12, 19 July 2011

UNIPV TEAM 2011

March
M T W T F S S
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31    

April
M T W T F S S
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4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30

May
M T W T F S S
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2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31

June
M T W T F S S
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30

July
M T W T F S S
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

August
M T W T F S S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31

September
M T W T F S S
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30

October
M T W T F S S
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31

JULY: WEEK 3

July, 11th

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E1-2 18 2.5 1 XbaI 1 PstI 2.5 25
E2-2 18 2.5 1 XbaI 1 PstI 2.5 25
E3-1 18 2.5 1 XbaI 1 PstI 2.5 25
E4-2 18 2.5 1 XbaI 1 PstI 2.5 25
E5-2 18 2.5 1 XbaI 1 PstI 2.5 25
E6-1 11 9.5 1 XbaI 1 PstI 2.5 25
E7-2 18 2.5 1 XbaI 1 PstI 2.5 25
E8-3 20.5 0 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_R0040</partinfo> 14.5 6 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_C0261</partinfo> 18 2.5 1 XbaI 1 PstI 2.5 25
<partinfo>BBa_I13507</partinfo> 10 10.5 1 XbaI 1 PstI 2.5 25
<partinfo>BBa_B0034</partinfo> 10 10.5 1 SpeI 1 PstI 2.5 25

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

In the afternoon gel electrophoresis was performed. 

                
 link alle 2 img dei gel con ling wiki

As shown, in figure all clones were positive, so we cut and purified the bands of interest.

After gel extraction, cut DNA was quantified:

Plasmid DNA (ng/μl)
E1 (X-P) 4.2
E2 (X-P) 3.8
E3 (X-P) 4.1
E4 (X-P) 4.2
E5 (X-P) 5.6
E6 (X-P) 7.8
E7 (X-P) 6.4
E8 (S-P) 3.4
<partinfo>BBa_C0261</partinfo> (X-P) 4.8
<partinfo>BBa_R0040</partinfo> (S-P) 7.9
<partinfo>BBa_B0034</partinfo> (S-P) 11.0
<partinfo>BBa_I13507</partinfo> (X-P) 3.9

Then ligations were performed in a final volume of 10 μl:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E9 <partinfo>BBa_B0030</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E10 <partinfo>BBa_B0031</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E11 <partinfo>BBa_B0032</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E12 <partinfo>BBa_B0034</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E13 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E2 (X-P) 6.5 1 1
E14 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E3 (X-P) 6.5 1 1
E15 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E4 (X-P) 6.5 1 1
E16 <partinfo>BBa_R0040</partinfo> (S-P) 2 <partinfo>BBa_C0261</partinfo> (X-P) 6 1 1


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