Team:Paris Liliane Bettencourt/Notebook/2011/08/13/

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== Cyrille ==
== Cyrille ==
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* To test the possibility of cloning in this plasmid
* To test the possibility of cloning in this plasmid
-
3 times 500 ng of the pHM3 plasmid is digested in E P for two hours using fast digest enzymes. As the fragment is short, they underdo a PCR purification to get rid of the small 43 bp interrior fragment.
+
3 times 500 ng of the pHM3 plasmid is digested in E P for 30 min
 +
 
 +
3 times 500ng of J04450 in pSB1C3 is digested using E P sites.  
 +
 
 +
The result is loaded on a gel. The and will undergo a gel purification.
 +
[[File:CP1311_gelextr_S27.jpg|thumb|center|Insert digested before cutting the bands]]
 +
[[File:CP1311_gelextr_S27_cut.jpg|thumb|center|Insert digested after cutting the bands]]
 +
 
 +
[[File:CP1311_gelextr_pHM3.jpg|thumb|center|plasmid digested before cutting the bands]]
 +
[[File:CP1311_gelextr_phm3_cut.jpg|thumb|center|plasmid digested after cutting the bands]]
 +
 
 +
After the gel extraction the yields where very low, so that it was in the noise of the tecan machine. So I decided to pool together the threee tubes of the same serie.
 +
Once mixed, vortexed and centrifucated, the concentration was mesures again. The tecan says the concentration is around 10 and 20 ng/mL depending on the tube and on the measure. This is sufficient to carry on.
 +
 
 +
The ligation was done using the T4 DNA ligase. 5 tubes where prepared as explain:
 +
 
 +
<table border="1">
 +
 
 +
<tr>
 +
<td>Tube</td>
 +
<td>1</td>
 +
<td>2</td>
 +
<td>3</td>
 +
<td>4</td></tr>
 +
<tr>
 +
<td>10x T4 buffer</td>
 +
<td>2µL</td>
 +
<td>2µL</td>
 +
<td>2µL</td>
 +
<td>2µL</td></tr>
 +
<tr>
 +
<td>T4 DNA ligase</td>
 +
<td>1µL</td>
 +
<td>1µL</td>
 +
<td>1µL</td>
 +
<td>1µL</td></tr>
 +
<tr>
 +
<td>DNA pHM3 EcoRI-</td>
 +
<td>2µL</td>
 +
<td>2µL</td>
 +
<td>5µL</td>
 +
<td>5µL</td></tr>
 +
<tr>
 +
<td>J04450 </td>
 +
<td>2µL</td>
 +
<td>10µL</td>
 +
<td>5µL</td>
 +
<td>10µL</td></tr>
 +
<tr>
 +
<td>H2O</td>
 +
<td>13µL</td>
 +
<td>5µL</td>
 +
<td>7µL</td>
 +
<td>2µL</td></tr>
 +
</table>
 +
 
 +
And the control
 +
<table>
 +
<tr>
 +
<td>10x T4 buffer</td>
 +
<td>2µL</td></tr>
 +
<tr>
 +
<td>T4 ligase</td>
 +
<td>5 µL</td></tr>
 +
<tr>
 +
<td>DNA pHM3</td>
 +
<td>5µL</td></tr>
 +
<tr>
 +
<td>H2O</td>
 +
<td>12µL</td></tr>
 +
</table>
 +
 
 +
It is incubated one hour at 22°C with some mixing from time to time, and then the totality of the product will be transformed in MH1 cells
 +
 
 +
== Kevin ==
 +
===Results of transformation ===
 +
 
 +
All the plates has grown with uniform cultur and no spots.
 +
Positive control (S27) are fluorescent, and not all the others, so digestion works.
 +
 
 +
We have to test if the transformation works.
-
3 times 500ng of J04450 in pSB1C3 is digested using E P sites. The result is loaded on a gel and will undergo a gel purification.
+
===Cultur of transformated cells to test===
 +
Cultur overnight from cells in the plate has been done.

Latest revision as of 16:10, 27 August 2011

Team IGEM Paris 2011

Contents

Cyrille

Adding the RFP biobrick in pHM3

We want to insert the biobrick J04450 into pHM3 for three reasons:

  • To have a negative control of digestion
  • To restore the EXSP biobrick format in the plasmid
  • To test the possibility of cloning in this plasmid

3 times 500 ng of the pHM3 plasmid is digested in E P for 30 min

3 times 500ng of J04450 in pSB1C3 is digested using E P sites.

The result is loaded on a gel. The and will undergo a gel purification.

Insert digested before cutting the bands
Insert digested after cutting the bands
plasmid digested before cutting the bands
plasmid digested after cutting the bands

After the gel extraction the yields where very low, so that it was in the noise of the tecan machine. So I decided to pool together the threee tubes of the same serie. Once mixed, vortexed and centrifucated, the concentration was mesures again. The tecan says the concentration is around 10 and 20 ng/mL depending on the tube and on the measure. This is sufficient to carry on.

The ligation was done using the T4 DNA ligase. 5 tubes where prepared as explain:

Tube 1 2 3 4
10x T4 buffer 2µL 2µL 2µL 2µL
T4 DNA ligase 1µL 1µL 1µL 1µL
DNA pHM3 EcoRI- 2µL 2µL 5µL 5µL
J04450 2µL 10µL 5µL 10µL
H2O 13µL 5µL 7µL 2µL

And the control

10x T4 buffer 2µL
T4 ligase 5 µL
DNA pHM3 5µL
H2O 12µL

It is incubated one hour at 22°C with some mixing from time to time, and then the totality of the product will be transformed in MH1 cells

Kevin

Results of transformation

All the plates has grown with uniform cultur and no spots. Positive control (S27) are fluorescent, and not all the others, so digestion works.

We have to test if the transformation works.

Cultur of transformated cells to test

Cultur overnight from cells in the plate has been done.

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