Team:UNIPV-Pavia/Calendar/July/settimana4

From 2011.igem.org

(Difference between revisions)
Line 81: Line 81:
   <tr>
   <tr>
       <td></html>E13-1<html></td>
       <td></html>E13-1<html></td>
-
       <td>??Insert??</td>
+
       <td>Insert</td>
       <td>6</td>
       <td>6</td>
       <td>14.5</td>
       <td>14.5</td>
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   <tr>
   <tr>
       <td></html>E16-1<html></td>
       <td></html>E16-1<html></td>
-
       <td>??Insert??</td>
+
       <td>Insert</td>
       <td>4</td>
       <td>4</td>
       <td>16.5</td>
       <td>16.5</td>
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   <tr>
   <tr>
       <td></html>E21-1<html></td>
       <td></html>E21-1<html></td>
-
       <td>??Insert??</td>
+
       <td>Insert</td>
       <td>7.5</td>
       <td>7.5</td>
       <td>13</td>
       <td>13</td>
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   <tr>
   <tr>
       <td></html>E22-2<html></td>
       <td></html>E22-2<html></td>
-
       <td>??Insert??</td>
+
       <td>Insert</td>
       <td>10</td>
       <td>10</td>
       <td>10.5</td>
       <td>10.5</td>
Line 412: Line 412:
<a name="July.2C_19th"></a><h2> <span class="mw-headline">July, 19th</span></h2>
<a name="July.2C_19th"></a><h2> <span class="mw-headline">July, 19th</span></h2>
<p>
<p>
 +
 +
<p>
 +
E13 was newly digested for the subsequent gel extraction.
 +
</p>
 +
 +
<center>
 +
<table border="1">
 +
    <tr>
 +
      <td><b>Plasmid</b></td>
 +
      <td><b>Kind</b></td>
 +
      <td><b>DNA (&mu;l)</b></td>
 +
      <td><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
 +
      <td><b>Enzyme 1 (&mu;l)</b></td>
 +
      <td><b>Enzyme 2 (&mu;l)</b></td>
 +
      <td><b>Buffer H (&mu;l)</b></td>
 +
      <td><b>Final Volume (&mu;l)</b></td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td></html>E13<html></td>
 +
      <td>Insert</td>
 +
      <td>6</td>
 +
      <td>14.5</td>
 +
      <td>1 Xbal</td>
 +
      <td>1 Pstl</td>
 +
      <td>2.5</td>
 +
      <td>25</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
 +
 +
<p>
 +
After electrophoresis, E13 insert was succesfully identified and gel extracted (not shown). Meanwhile, sufficient amount of buffer 1 and buffer 2 for MGZ1 competent cell preparation protocol were prepared. E13 DNA was then quantified:
 +
</p>
 +
 +
<center>
 +
<table border="1">
 +
    <tr>
 +
      <td><b>Plasmid</b></td>
 +
      <td><b>DNA (ng/&mu;l)</b></td>
 +
  </tr>
 +
 +
  <tr>
 +
      <td>E13 (X-P)</td>
 +
      <td>43</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
 +
 +
<p>
 +
After E13 digestion (E-P), its ligation was performed in a final volume of 10 &mu;l:
 +
</p>
 +
 +
<center>
 +
<table border="1">
 +
    <tr>
 +
      <td><b>Ligation Name</b></td>
 +
      <td><b>Vector</b></td>
 +
      <td><b>Vector volume (&mu;l)</b></td>
 +
      <td><b>Insert</b></td>
 +
      <td><b>Insert volume (&mu;l)</b></td>
 +
      <td><b>Buffer (&mu;l)</b></td>
 +
      <td><b>T4 Ligase (&mu;l)</b></td>
 +
  </tr>
 +
 +
    <tr>
 +
      <td><b>E28</b></td>
 +
      <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
 +
      <td>6</td>
 +
      <td>E9 (X-P)</td>
 +
      <td>2</td>
 +
      <td>1</td>
 +
      <td>1</td>
 +
  </tr>
 +
 +
</table>
 +
</center>
 +
 +
<p>
 +
Ligations were incubated ON at 16°C.
 +
</p>

Revision as of 11:03, 21 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
        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

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 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
<partinfo>BBa_I13521</partinfo> Insert 12.5 8 1 EcoRl 1 Pstl 2.5 25
<partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo> Vector 20.5 0 1 EcoRl 1 Pstl 2.5 25

Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols. Moreover BBa_I13521 plasmid purification and quantification were carried out:

In the afternoon gel electrophoresis was performed.

Plasmid DNA (ng/μl)
<partinfo>BBa_I13521</partinfo> 37.3

2 righe sopra inserire l' immagine

As shown in figure, all clones were positive (apart from E13 run where it is possible to recognize only a band at approximately 2800 bp, corresponding to the undigested plasmid), so we cut and purified the bands of interest. Cells harbouring E13 were again inoculated in 5 ml LB + Amp.

E13 was newly then digested with restriction endonucleases:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
<partinfo>BBa_I13521</partinfo> Insert 1 19.5 1 EcoRl 1 Pstl 2.5 25

After gel extraction, cut DNA was quantified:

Plasmid DNA (ng/μl)
E9 (X-P) 5.9
E10 (X-P) 4.2
E11 (X-P) 5.2
E12 (X-P) 5.2
E16 (E-P) 6.4
E21 (E-P) 5.8
E22 (E-P) 6.5
E23 (E-P) 4.6
<partinfo>BBa_I13521</partinfo> 6.1
<partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo> 2.1

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)
E24 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E9 (X-P) 6.5 1 1
E25 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E10 (X-P) 6.5 1 1
E26 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E11 (X-P) 6.5 1 1
E27 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E12 (X-P) 6.5 1 1
E31 <partinfo>pSB4C5</partinfo> (E-P) 2.5 E16 (E-P) 5.5 1 1
E32 <partinfo>pSB4C5</partinfo> (E-P) 2 E21 (E-P) 6 1 1
E33 <partinfo>pSB4C5</partinfo> (E-P) 2 E22 (E-P) 6 1 1
E34 <partinfo>pSB4C5</partinfo> (E-P) 6.5 E23 (E-P) 1.5 1 1
E35 <partinfo>pSB4C5</partinfo> (E-P) 2 <partinfo>BBa_I13521</partinfo>(E-P) 6.5 1 1
E36 <partinfo>pSB4C5</partinfo> (E-P) 1 <partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo>(E-P) 7 1 1

Ligations were incubated ON at 16°C.

July, 19th

E13 was newly digested for the subsequent gel extraction.

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E13 Insert 6 14.5 1 Xbal 1 Pstl 2.5 25

After electrophoresis, E13 insert was succesfully identified and gel extracted (not shown). Meanwhile, sufficient amount of buffer 1 and buffer 2 for MGZ1 competent cell preparation protocol were prepared. E13 DNA was then quantified:

Plasmid DNA (ng/μl)
E13 (X-P) 43

After E13 digestion (E-P), its ligation was performed in a final volume of 10 μl:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E28 <partinfo>pSB4C5</partinfo> (E-P) 6 E9 (X-P) 2 1 1

Ligations were incubated ON at 16°C.

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