Team:UNIPV-Pavia/Calendar/July/settimana4

From 2011.igem.org

(Difference between revisions)
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Digestions of previously purified plasmids were performed for ligations:
Digestions of previously purified plasmids were performed for ligations:
</p>
</p>
-
 
-
<p>CAMBIARE OGGETTI TABELLA</p>
 
<center>
<center>
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   <tr>
   <tr>
-
       <td></html><partinfo>BBa_C0061</partinfo><html></td>
+
       <td></html>E10-1<html></td>
       <td>Insert</td>
       <td>Insert</td>
-
       <td>13.2</td>
+
       <td>10</td>
-
       <td>7.3</td>
+
       <td>10.5</td>
-
       <td>1 XbaI</td>
+
       <td>1 Xbal</td>
-
       <td>1 PstI</td>
+
       <td>1 Pstl</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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   <tr>
   <tr>
-
       <td></html><partinfo>BBa_K081022</partinfo><html></td>
+
       <td></html>E11-1<html></td>
       <td>Insert</td>
       <td>Insert</td>
-
       <td>15.7</td>
+
       <td>4</td>
-
       <td>4.8</td>
+
       <td>16.5</td>
-
       <td>1 EcoRI</td>
+
       <td>1 Xbal</td>
-
       <td>1 PstI</td>
+
       <td>1 Pstl</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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   <tr>
   <tr>
-
       <td></html><partinfo>BBa_B0030</partinfo><html></td>
+
       <td></html>E12-1<html></td>
-
       <td>Vector</td>
+
       <td>Insert</td>
-
       <td>13.2</td>
+
       <td>10</td>
-
       <td>7.3</td>
+
       <td>10.5</td>
-
       <td>1 SpeI</td>
+
       <td>1 Xbal</td>
       <td>1 PstI</td>
       <td>1 PstI</td>
       <td>2.5</td>
       <td>2.5</td>
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   <tr>
   <tr>
-
       <td></html><partinfo>BBa_B0031</partinfo><html></td>
+
       <td></html>E13-1<html></td>
-
       <td>Vector</td>
+
       <td>??Insert??</td>
-
       <td>12.4</td>
+
       <td>6</td>
-
       <td>8.1</td>
+
       <td>14.5</td>
-
       <td>1 SpeI</td>
+
       <td>1 EcoRl</td>
-
       <td>1 PstI</td>
+
       <td>1 Pstl</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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   <tr>
   <tr>
-
       <td></html><partinfo>BBa_B0032</partinfo><html></td>
+
       <td></html>E16-1<html></td>
-
       <td>Vector</td>
+
       <td>??Insert??</td>
-
       <td>9.5</td>
+
       <td>4</td>
-
       <td>11</td>
+
       <td>16.5</td>
-
       <td>1 SpeI</td>
+
       <td>1 EcoRl</td>
-
       <td>1 PstI</td>
+
       <td>1 Pstl</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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   <tr>
   <tr>
-
       <td></html><partinfo>BBa_B0015</partinfo><html></td>
+
       <td></html>E21-1<html></td>
-
       <td>Vector</td>
+
       <td>??Insert??</td>
-
       <td>7.9</td>
+
       <td>7.5</td>
-
       <td>12.6</td>
+
       <td>13</td>
-
       <td>1 EcoRI</td>
+
       <td>1 EcoRl</td>
-
       <td>1 XbaI</td>
+
       <td>1 Xbal</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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   <tr>
   <tr>
-
       <td></html><partinfo>pSB4C5</partinfo><html></td>
+
       <td></html>E22-2<html></td>
-
       <td>Vector</td>
+
       <td>??Insert??</td>
-
       <td>3</td>
+
       <td>10</td>
-
       <td>17.5</td>
+
       <td>10.5</td>
-
       <td>1 EcoRI</td>
+
       <td>1 EcoRl</td>
-
       <td>1 PstI</td>
+
       <td>1 Pstl</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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   <tr>
   <tr>
-
       <td></html><partinfo>BBa_I13501</partinfo><html></td>
+
       <td></html>I13521<html></td>
       <td>Insert</td>
       <td>Insert</td>
-
       <td>7.2</td>
+
       <td>12.5</td>
-
       <td>13.3</td>
+
      <td>8</td>
-
       <td>1 XbaI</td>
+
      <td>1 EcoRl</td>
-
       <td>1 PstI</td>
+
      <td>1 Pstl</td>
 +
      <td>2.5</td>
 +
       <td>25</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
      <td></html>R0040-J61002<html></td>
 +
      <td>Vector</td>
 +
      <td>20.5</td>
 +
      <td>0</td>
 +
       <td>1 EcoRl</td>
 +
       <td>1 Pstl</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>

Revision as of 09:31, 21 July 2011

UNIPV TEAM 2011

March
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April
M T W T F S S
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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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23 24 25 26 27 28 29
30 31

June
M T W T F S S
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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
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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
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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
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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 4

July, 18th

Digestions of previously purified plasmids were performed for ligations:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E9-2 Insert 11.5 9 1 Xbal 1 Pstl 2.5 25
E10-1 Insert 10 10.5 1 Xbal 1 Pstl 2.5 25
E11-1 Insert 4 16.5 1 Xbal 1 Pstl 2.5 25
E12-1 Insert 10 10.5 1 Xbal 1 PstI 2.5 25
E13-1 ??Insert?? 6 14.5 1 EcoRl 1 Pstl 2.5 25
E16-1 ??Insert?? 4 16.5 1 EcoRl 1 Pstl 2.5 25
E21-1 ??Insert?? 7.5 13 1 EcoRl 1 Xbal 2.5 25
E22-2 ??Insert?? 10 10.5 1 EcoRl 1 Pstl 2.5 25
I13521 Insert 12.5 8 1 EcoRl 1 Pstl 2.5 25
R0040-J61002 Vector 20.5 0 1 EcoRl 1 Pstl 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.

2 righe sopra inserire l' immagine

As shown, in figure all clones were positive (apart from <partinfo>BBa_I13501</partinfo> run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring <partinfo>BBa_I13501</partinfo> were again inoculated in 5 ml LB + Amp.

After gel extraction, cut DNA was quantified:

Plasmid DNA (ng/μl)
<partinfo>BBa_C0060</partinfo> (E-S) 3.9
<partinfo>BBa_C0061</partinfo> (X-P) 4.1
<partinfo>BBa_K081022</partinfo> (E-P) 4.4
<partinfo>BBa_B0030</partinfo> (S-P) 10.3
<partinfo>BBa_B0031</partinfo> (S-P) 11.8
<partinfo>BBa_B0032</partinfo> (S-P) 10.1
<partinfo>BBa_B0015</partinfo> (E-X) 12
<partinfo>pSB4C5</partinfo> (E-P) 10.7

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)
E1 <partinfo>BBa_B0015</partinfo> (E-X) 1.5 <partinfo>BBa_C0060</partinfo> (E-S) 6.5 1 1
E2 <partinfo>BBa_B0030</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E3 <partinfo>BBa_B0031</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E4 <partinfo>BBa_B0032</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E8 <partinfo>pSB4C5</partinfo> (E-P) 1.5 <partinfo>BBa_K081022</partinfo> (E-P) 6.5 1 1

Ligations were incubated ON at 16°C.

July, 19th

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Laboratory for Biomedical Informatics | Centre of Tissutal Engineering

University of Pavia

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