Team:UNIPV-Pavia/Calendar/July/settimana5

From 2011.igem.org

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<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<a name="July.2C_26th"></a><h2> <span class="mw-headline">July, 26th</span></h2>
<a name="July.2C_26th"></a><h2> <span class="mw-headline">July, 26th</span></h2>
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_26_07_2011_E36_S-P.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d4/UNIPV_26_07_2011_E36_S-P.png" class="thumbimage" height="80%" width="80%"></a> <div class="thumbcaption">Small size gel</div></div></div></div>
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<div class="center"><div class="thumb tnone"><div class="thumbinner" style="width: 400px;"><a href="/File:UNIPV_26_07_2011_E36_S-P.png" class="image"><img alt="" src="https://static.igem.org/mediawiki/2011/d/d4/UNIPV_26_07_2011_E36_S-P.png" class="thumbimage" height="80%" width="80%"></a><div class="thumbcaption">Small size gel</div></div></div></div>
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E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 μl of MGZ1 competent cells according to protocols. Moreover a plasmid containing <a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13501">BBa_B0032</a> was transformed in order to test the transformation efficiency. Plates were incubated ON at 37°C.
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E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 &mu;l of MGZ1 competent cells according to protocols. Moreover 4ng of purified <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0032">BBa_B0032</a> DNA were transformed in MGZ1 in order to test the transformation efficiency. Plates were incubated ON at 37°C.<br>
 +
BMR Genomics sent results of parts sequencing; E17-2, E18-2, E31-1, E32-2, E33-2, E34-1, E35-2 were OK while E28-1 sequence was wrong.
</p>
</p>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<div align="right"><small><a href="#indice" title="">^top</a></small></div>
<a name="July.2C_28th"></a><h2> <span class="mw-headline">July, 28th</span></h2>
<a name="July.2C_28th"></a><h2> <span class="mw-headline">July, 28th</span></h2>
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<p>
<p>
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Colonies grew in every plate. In fact E17-2, E18-2, E19-2 and E20-2, E37, E38, E39 and E40 showed grown colonies. We picked two colonies for each plate and inoculate them in 5 ml of LB + Cm 12.5. As regards to E41 and E42 parts, we resolved to let them still grow; in the late morning we picked and inoculate two colonies for both plates.<br>
 +
Plate with <a href="http://partsregistry.org/wiki/index.php/Part:BBa_B0032">BBa_B0032</a> showed 9 colonies; the corresponding efficiency results in 2250 colonies/&mu;g of DNA (very low, we need to prepare new competent MGZ1).
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</p>
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<div align="right"><small><a href="#indice" title="">^top</a></small></div>
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<a name="July.2C_29th"></a><h2> <span class="mw-headline">July, 29th</span></h2>
<p>
<p>
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Colonies grew in every plate. Infact both 17-2, E18-2, E19-2 and E20-2 parts, whose DNA was recovered after MiniPrep extraction kit, both E37, E38, E39 and E40 parts showed grown colonies. We picked two colonies from all parts. As regards to E41 and E42 parts, we resolved to let them still grow.<br>
+
Plasmid purification was carried out on over-night grown cultures (E41-1 did not grow!):
-
Plate with <a href="http://partsregistry.org/wiki/index.php/Part:BBa_I13501">BBa_B0032</a> showed 9 colonies. The corresponding efficiency results 9/4 [1/ng] = 2250 [1/&mu;l] 
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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>
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  </tr>
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  <tr>
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      <td>E37-1</td>
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      <td>16.6</td>
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  </tr>
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  <tr>
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      <td>E37-2</td>
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      <td>16.9</td>
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  </tr>
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  <tr>
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      <td>E38-1</td>
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      <td>28.4</td>
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  </tr>
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  <tr>
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      <td>E38-2</td>
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      <td>15.0</td>
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  </tr>
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  <tr>
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      <td>E39-1</td>
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      <td>15.0</td>
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  </tr>
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  <tr>
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      <td>E39-2</td>
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      <td>14.5</td>
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  </tr>
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  <tr>
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      <td>E40-1</td>
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      <td>17.1</td>
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  </tr>
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  <tr>
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      <td>E40-2</td>
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      <td>38.9</td>
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  </tr>
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  <tr>
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      <td>E41-2</td>
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      <td>13.7</td>
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  </tr>
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  <tr>
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      <td>E42-1</td>
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      <td>22.7</td>
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  </tr>
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  <tr>
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      <td>E42-2</td>
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      <td>28</td>
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  </tr>
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</table>
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</center>
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<p>
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A 12x mix was prepared for screening digstion:
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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>H<small><sub>2</sub></small>O (μl)</b></td>
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      <td><b>Enzyme 1 (μl)</b></td>
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      <td><b>Enzyme 2 (μl)</b></td>
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      <td><b>Buffer H (μl)</b></td>
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  </tr>
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<tr>
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      <td>126</td>
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      <td>12 EcoRI</td>
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      <td>12 PstI</td>
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      <td>30</td>
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  </tr>
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</table>
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</center>
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<p>
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For each digestion 10 &mu;l of purified DNA were used. Meanwhile a medium size gel was prepared.<br>
 +
In the afternoon gel electrophoresis was carried out:
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</p>
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<table border="0" width="100%" class="menu">
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<tr>
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<td align="left"><a href="/Team:UNIPV-Pavia/Calendar/July/settimana4" title="Team:UNIPV-Pavia/Calendar/JuLY/settimana4"> Previous week</a></td>
 +
<td align="right"><a href="/Team:UNIPV-Pavia/Calendar/August/settimana1" title="Team:UNIPV-Pavia/Calendar/August/settimana1"> Next week</a></td>
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</tr>
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</table>
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</html>
</html>
{{end}}
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Revision as of 13:26, 30 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 5

July, 25th

Red colonies were observed from E36 plate, suggesting that the transformation was successful. A colony was picked form the plate.

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)
E24-2 Insert 13 7.5 1 Xbal 1 Pstl 2.5 25
E25-1 Insert 13.5 7 1 Xbal 1 Pstl 2.5 25
E26-2 Insert 14.5 6 1 Xbal 1 Pstl 2.5 25
E27-2 Insert 12.5 8 1 Xbal 1 PstI 2.5 25

Reactions were incubated at 37°C for three hours while a small size agarose gel was prepared according to protocols.
In the afternoon gel electrophoresis was performed:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
E24-2 (E-P) 7.4
E25-1 (E-P) 8.4
E26-2 (E-P) 3.2
E27-2 (E-P) 6.3

Ligations were performed:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E37 pSB4C5 (E-P) 2 E24-2 (E-P) 6 1 1
E38 pSB4C5 (E-P) 2.5 E25-1 (E-P) 5.5 1 1
E39 pSB4C5 (E-P) 1 E26-2 (E-P) 7 1 1
E40 pSB4C5 (E-P) 2 E27-2 (E-P) 6 1 1

Inoculum of E17-2, E18-2, E19-2 or E20-2 to amplify DNA and transfer it into MGZ1; refill for E36 liquid culture.
Glycerol stock fpr E36 was prepared
250 ml of LB + Cm 12.5 were prepared.

July, 26th

Plasmids containing E17-2, E18-2, E19-2, E20-2 and E36 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:

Plasmid DNA (ng/μl)
E17-2 20.3
E18-2 18.6
E19-2 17.3
E20-2 13.2
E36 17.8

Digestion of E36 was performed for ligations:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E36 Vector 20.5 0 1 Spel 1 Pstl 2.5 25

The sample was incubated at 37°C for three hours while a small-size agarose gel was prepared; in the afteroon gel electrophoresis was carried out:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
E36 (S-P) 3.5

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)
E41 E36 (S-P) 4 E3-1 (X-P) 4 1 1
E42 E36 (S-P) 3.5 E4-2 (X-P) 4.5 1 1

Ligations were incubated at 16°C ON.

July, 27th

E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 μl of MGZ1 competent cells according to protocols. Moreover 4ng of purified BBa_B0032 DNA were transformed in MGZ1 in order to test the transformation efficiency. Plates were incubated ON at 37°C.
BMR Genomics sent results of parts sequencing; E17-2, E18-2, E31-1, E32-2, E33-2, E34-1, E35-2 were OK while E28-1 sequence was wrong.

July, 28th

Colonies grew in every plate. In fact E17-2, E18-2, E19-2 and E20-2, E37, E38, E39 and E40 showed grown colonies. We picked two colonies for each plate and inoculate them in 5 ml of LB + Cm 12.5. As regards to E41 and E42 parts, we resolved to let them still grow; in the late morning we picked and inoculate two colonies for both plates.
Plate with BBa_B0032 showed 9 colonies; the corresponding efficiency results in 2250 colonies/μg of DNA (very low, we need to prepare new competent MGZ1).

July, 29th

Plasmid purification was carried out on over-night grown cultures (E41-1 did not grow!):

Plasmid DNA (ng/μl)
E37-1 16.6
E37-2 16.9
E38-1 28.4
E38-2 15.0
E39-1 15.0
E39-2 14.5
E40-1 17.1
E40-2 38.9
E41-2 13.7
E42-1 22.7
E42-2 28

A 12x mix was prepared for screening digstion:

H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl)
126 12 EcoRI 12 PstI 30

For each digestion 10 μl of purified DNA were used. Meanwhile a medium size gel was prepared.
In the afternoon gel electrophoresis was carried out:

Previous week Next week

Laboratory for Biomedical Informatics | Centre of Tissutal Engineering

University of Pavia

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