Team:UNIPV-Pavia/Calendar/July/settimana3

From 2011.igem.org

(Difference between revisions)
(Created page with "{{maincal}} <html> <h2 class="art-postheader"> JULY: WEEK 2 </h2> <div class="cleared"></div> <div class="art-postcontent"> <p style="text-align:left;"> <p><a name="indice"/> ...")
Line 2: Line 2:
<html>
<html>
  <h2 class="art-postheader">
  <h2 class="art-postheader">
-
JULY: WEEK 2
+
JULY: WEEK 3
</h2>
</h2>
<div class="cleared"></div>
<div class="cleared"></div>
Line 11: Line 11:
<p><a name="indice"/>
<p><a name="indice"/>
</p>
</p>
-
<a name="July.2C_4th"></a><h2> <span class="mw-headline">July, 4th</span></h2>
+
<a name="July.2C_11th"></a><h2> <span class="mw-headline">July, 11th</span></h2>
<p>
<p>
Line 18: Line 18:
</p>
</p>
 +
<p>CAMBIARE OGGETTI TABELLA</p>
<center>
<center>
Line 142: Line 143:
<p>
<p>
-
Reactions were incubated at 37°C for three hours while a small-size and a medium-size agarose gel were prepared according to protocols.
+
Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.
</p>
</p>
Line 149: Line 150:
</p>
</p>
-
</html>[[Image:UNIPV_04_07_SSgel.png|frame|center|300px|Small size gel]]
+
</html>
-
[[Image:UNIPV_04_07_MSgel.png|frame|center|300px|Medium size gel]]<html>
+
 +
<html>
 +
<p>2 righe sopra inserire l' immagine</p>
<p>
<p>
Line 297: Line 299:
</p>
</p>
-
<a name="July.2C_5th"></a><h2> <span class="mw-headline">July, 5th</span></h2>
+
<a name="July.2C_19th"></a><h2> <span class="mw-headline">July, 19th</span></h2>
<p>
<p>
-
<p>
 
-
T4 Ligase was inactivated, heating ligations at 65°C for 10 minutes; ligations were stored at -20°C. Plasmid purification was done again for </html><partinfo>BBa_I13501</partinfo><html> and purified DNA was quantified:
 
-
</p>
 
-
<center>
 
-
<table border="1">
 
-
    <tr>
 
-
      <td><b>Plasmid</b></td>
 
-
      <td><b>DNA (ng/&mu;l)</b></td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td></html><partinfo>BBa_I13501</partinfo><html></td>
 
-
      <td>97.2</td>
 
-
  </tr>
 
-
</table>
 
-
</center>
 
-
 
-
<p>
 
-
</html><partinfo>BBa_I13501</partinfo><html> was then digested with restriction endonucleases:
 
-
</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><partinfo>BBa_I13501</partinfo><html></td>
 
-
      <td>Insert</td>
 
-
      <td>10.5</td>
 
-
      <td>10</td>
 
-
      <td>1 XbaI</td>
 
-
      <td>1 PstI</td>
 
-
      <td>2.5</td>
 
-
      <td>25</td>
 
-
  </tr>
 
-
</table>
 
-
</center>
 
-
 
-
<p>
 
-
According to protocols the reaction was incubated at 37°C for 3 hours.<br>
 
-
60 ml of 80% glycerol were prepared mixing 48 ml of 100% glycerol with 12 ml of ddH<small><sub>2</small></sub>O.<br>
 
-
Gel electrophoresis was done for </html><partinfo>BBa_I13501</partinfo><html> digestion:
 
-
</p>
 
-
 
-
</html>[[Image:UNIPV_05_07_SSgel.png|frame|center|300px|Small size gel]]<html>
 
-
 
-
<p>
 
-
Cut DNA was gel-extracted:
 
-
</p>
 
-
 
-
<center>
 
-
<table border="1">
 
-
    <tr>
 
-
      <td><b>Plasmid</b></td>
 
-
      <td><b>DNA (ng/&mu;l)</b></td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td></html><partinfo>BBa_I13501</partinfo><html> (X-P)</td>
 
-
      <td>2.6</td>
 
-
  </tr>
 
-
</table>
 
-
</center>
 
-
 
-
<p>
 
-
Further ligations were prepared and incubated ON at 16°C:
 
-
</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>E5</b></td>
 
-
      <td></html><partinfo>BBa_B0030</partinfo><html> (S-P)</td>
 
-
      <td>1</td>
 
-
      <td></html><partinfo>BBa_I13501</partinfo><html> (X-P)</td>
 
-
      <td>7</td>
 
-
      <td>1</td>
 
-
      <td>1</td>
 
-
  </tr>
 
-
 
-
 
-
    <tr>
 
-
      <td><b>E6</b></td>
 
-
      <td></html><partinfo>BBa_B0031</partinfo><html> (S-P)</td>
 
-
      <td>1</td>
 
-
      <td></html><partinfo>BBa_I13501</partinfo><html> (X-P)</td>
 
-
      <td>7</td>
 
-
      <td>1</td>
 
-
      <td>1</td>
 
-
  </tr>
 
-
 
-
    <tr>
 
-
      <td><b>E7</b></td>
 
-
      <td></html><partinfo>BBa_B0032</partinfo><html> (S-P)</td>
 
-
      <td>1</td>
 
-
      <td></html><partinfo>BBa_I13501</partinfo><html> (X-P)</td>
 
-
      <td>7</td>
 
-
      <td>1</td>
 
-
      <td>1</td>
 
-
  </tr>
 
-
 
-
</table>
 
-
</center>
 
-
 
-
<a name="July.2C_6th"></a><h2> <span class="mw-headline">July, 6th</span></h2>
 
-
<p>
 
-
</html><partinfo>BBa_C0261</partinfo><html> was resuspended from iGEM 2011 kit distribution, Plate 1, well 14C in 15 &mu;l of ddH<small><sub>2</sub></small>O; </html><partinfo>BBa_C0261</partinfo><html>, E1, E2, E3, E4, E5, E6, E7, and E8 were transformed in 100 &mu;l of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.<br>
 
-
500 ml of LB without antibiotic were prepared.
 
-
</p>
 
-
 
-
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 
-
 
-
<a name="July.2C_7th"></a><h2> <span class="mw-headline">July, 7th</span></h2>
 
-
<p>
 
-
All plates showed a lot of colonies, except for E-8 (5 colonies); two colonies for E1, E2, E3, E4, E5, E6, E7 plates and three colonies for E8 plate were picked and inoculated in 10 &mu;l LB for inoculum and screening PCR.<br>
 
-
A 20x mix was prepared for PCR reaction:
 
-
</p>
 
-
 
-
 
-
<center>
 
-
<table border="1">
 
-
    <tr>
 
-
      <td><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
 
-
      <td><b>Buffer 10x (&mu;l)</b></td>
 
-
      <td><b>MgCl<small><sub>2</sub></small> (&mu;l)</b></td>
 
-
      <td><b>VF2 (</html><partinfo>BBa_G00100</partinfo><html>) &mu;l</b></td>
 
-
      <td><b>VR (</html><partinfo>BBa_G00101</partinfo><html>) &mu;l</b></td>
 
-
      <td><b>dNTPs (&mu;l)</b></td>
 
-
      <td><b>Taq polymerase (&mu;l)</b></td>
 
-
  </tr>
 
-
 
-
    <tr>
 
-
      <td>360</td>
 
-
      <td>50</td>
 
-
      <td>20</td>
 
-
      <td>10</td>
 
-
      <td>10</td>
 
-
      <td>10</td>
 
-
      <td>20</td>
 
-
  </tr>
 
-
 
-
</table>
 
-
</center>
 
-
 
-
<p>
 
-
24 &mu;l were aliquoted in each tube, together with 1 &mu;l of each liquid culture. PCR cycles parameters were set as follows:
 
-
<ul type="circle">
 
-
<li> 94°C 30 seconds (denaturing)
 
-
<li> 60°C 1 minute (annealing)
 
-
<li> 72°C 1 minute and 20 seconds (elongation)
 
-
</ul>
 
-
35 cycles were performed.<br>
 
-
Liquid cultures were then inoculated in 800 &mu;l LB with the proper antibiotic.<br>
 
-
A small size and a medium size agarose gel were prepared.
 
-
</p>
 
-
 
-
<p>
 
-
After the PCR, gel run was carried out on the samples:
 
-
</p>
 
-
 
-
 
-
</html>[[Image:UNIPV_07_07_MSgel.png|frame|center|300px|Medium size gel]]
 
-
[[Image:UNIPV_07_07_SSgel.png|frame|center|300px|Small size gel]]<html>
 
-
 
-
<p>
 
-
Except for E2-1, E4-1 and E7-1, other band lengths were correct; 750 &mu;l of E1-2, E2-2, E3-1, E4-2, E5-2, E6-1, E7-2 and E8-3 liquid cultures were used to prepare glycerol stocks while the remaining 50 &mu;l were refilled to 5 ml for plasmid purification and incubated at 37°C 220 rpm. </html><partinfo>BBa_R0040</partinfo><html>, </html><partinfo>BBa_C0261</partinfo><html> and </html><partinfo>BBa_I13521</partinfo><html> were also inoculated.<br>
 
-
In the late afternoon the team met to talk about the wet-lab activity, the abstract and the bio-safety section.
 
-
</p>
 
-
 
-
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 
-
 
-
<a name="July.2C_8th"></a><h2> <span class="mw-headline">July, 8th</span></h2>
 
-
 
-
<p>
 
-
Cultures were saturated; plasmid purification was carried out:
 
-
</p>
 
-
 
-
 
-
 
-
<center>
 
-
<table border="1">
 
-
    <tr>
 
-
      <td><b>Plasmid</b></td>
 
-
      <td><b>DNA (ng/&mu;l)</b></td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td>E1-2</td>
 
-
      <td>64.1</td>
 
-
  </tr>
 
-
 
-
 
-
  <tr>
 
-
      <td>E2-2</td>
 
-
      <td>56.2</td>
 
-
  </tr>
 
-
 
-
 
-
  <tr>
 
-
      <td>E3-1</td>
 
-
      <td>53.5</td>
 
-
  </tr>
 
-
 
-
 
-
  <tr>
 
-
      <td>E4-2</td>
 
-
      <td>67.2</td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td>E5-2</td>
 
-
      <td>73.5</td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td>E6-1</td>
 
-
      <td>92.2</td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td>E7-2</td>
 
-
      <td>67.6</td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td>E8-3</td>
 
-
      <td>25.3</td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td></html><partinfo>BBa_R0040</partinfo><html></td>
 
-
      <td>48.1</td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td></html><partinfo>BBa_C0261</partinfo><html></td>
 
-
      <td>61.2</td>
 
-
  </tr>
 
-
 
-
  <tr>
 
-
      <td></html><partinfo>BBa_I13521</partinfo><html></td>
 
-
      <td>79.3</td>
 
-
  </tr>
 
-
 
-
</table>
 
-
</center>
 
-
 
-
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 
-
 
-
 
-
<table border="0" width="100%" class="menu">
 
-
<tr>
 
-
<td align="left"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana1" title="Team:UNIPV-Pavia/Calendar/JuLY/settimana1"> Previous week</a></td>
 
-
<td align="right"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana3" title="Team:UNIPV-Pavia/Calendar/July/settimana3"> Next week</a></td>
 
-
 
-
</tr>
 
-
</table>
 
</html>
</html>
{{end}}
{{end}}

Revision as of 00:20, 19 July 2011

UNIPV TEAM 2011

March
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    

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
            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

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

Digestions of previously purified plasmids were performed for ligations:

CAMBIARE OGGETTI TABELLA

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
<partinfo>BBa_C0060</partinfo> Insert 16.5 4 1 EcoRI 1 SpeI 2.5 25
<partinfo>BBa_C0061</partinfo> Insert 13.2 7.3 1 XbaI 1 PstI 2.5 25
<partinfo>BBa_K081022</partinfo> Insert 15.7 4.8 1 EcoRI 1 PstI 2.5 25
<partinfo>BBa_B0030</partinfo> Vector 13.2 7.3 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0031</partinfo> Vector 12.4 8.1 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0032</partinfo> Vector 9.5 11 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0015</partinfo> Vector 7.9 12.6 1 EcoRI 1 XbaI 2.5 25
<partinfo>pSB4C5</partinfo> Vector 3 17.5 1 EcoRI 1 PstI 2.5 25
<partinfo>BBa_I13501</partinfo> Insert 7.2 13.3 1 XbaI 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.

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

Laboratory for Biomedical Informatics | Centre of Tissutal Engineering

University of Pavia

Retrieved from "http://2011.igem.org/Team:UNIPV-Pavia/Calendar/July/settimana3"