Team:Exeter/lab book/3gip/wk4

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         </p>
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         <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk11"; style="color:#1d1d1b">17th - 21st September</a>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk11"; style="color:#1d1d1b">17th - 21st September</a>
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        <a href="https://2012.igem.org/Team:Exeter/Results/inducible"; style="color:#e30614" target="_blank"><font size="3"><b>Results</b></font></a>
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</font>
       </font>
       </font>
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     </div>
     <!--End Project Division Week Hyperlinks-->
     <!--End Project Division Week Hyperlinks-->
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   <td width="850" height="250">
   <!------------INSERT WEEKLY IMAGE HERE------------>
   <!------------INSERT WEEKLY IMAGE HERE------------>
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     <p><b><u>Monday 30th July</u></b></p>
     <p><b><u>Monday 30th July</u></b></p>
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
-
<p>•<u> Adding cultures</u></p>   
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Transferring colonies to liquid medium</u></a></p>   
<p>Cells transformed with BBa_I0500 were added into liquid medium and incubated overnight. </p>
<p>Cells transformed with BBa_I0500 were added into liquid medium and incubated overnight. </p>
<p>Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic. </p><br>
<p>Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic. </p><br>
Line 118: Line 122:
<p><b><u>Tuesday 31th July</u></b></p>
<p><b><u>Tuesday 31th July</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>•<u> Mini-Prepping</u></p>
+
<p>• <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Miniprepping</u></a></p>
<p>BBa_I0500</p>
<p>BBa_I0500</p>
<p>BBa_I0500 was nanodropped and recorded poor concentrations. </p><br>
<p>BBa_I0500 was nanodropped and recorded poor concentrations. </p><br>
 +
<p>•<u>Gel Electrophoresis</p></u>
<p>•<u>Gel Electrophoresis</p></u>
<p>Run to check fragment sizes of BBa_I0500 after digestion using the EcoR1 and Pst1 enzymes</p>
<p>Run to check fragment sizes of BBa_I0500 after digestion using the EcoR1 and Pst1 enzymes</p>
<p>Gel Electrophoresis showed a faint band at the correct size. </p><br>
<p>Gel Electrophoresis showed a faint band at the correct size. </p><br>
 +
<p>•<u> Sequencing</u></p>   
<p>•<u> Sequencing</u></p>   
<p>BBa_K094120_ BBa_B0034 was sent off for sequencing using the primers VF2 and VR. </p><br>
<p>BBa_K094120_ BBa_B0034 was sent off for sequencing using the primers VF2 and VR. </p><br>
-
<p>•<u>3A assembly:</u></p>   
+
 
 +
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A Assembly</u></a></p>   
<p>BBa_I0500 + BBa_B0034</p>
<p>BBa_I0500 + BBa_B0034</p>
<p>Performed with the NEB protocol but using the Linearized Plasmid Protocol from the parts registry. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. As described in the Linearized Plasmid Protocol, a mastermix was made and 4μl of this was added to 4μl of destination plasmid for the digestion. </p><br>
<p>Performed with the NEB protocol but using the Linearized Plasmid Protocol from the parts registry. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. As described in the Linearized Plasmid Protocol, a mastermix was made and 4μl of this was added to 4μl of destination plasmid for the digestion. </p><br>
 +
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
-
<p>•<u>Transformation:</u></p>   
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>Transformation</u></a></p>   
<p>BBa_I0500 + BBa_B0034</p>
<p>BBa_I0500 + BBa_B0034</p>
<p>Using Invitrogen TOP10 competent cells. </p>
<p>Using Invitrogen TOP10 competent cells. </p>
-
<p>Spread on two kanamycin plates with 20μl and 100μls respectively. </p>  
+
<p>Spread on two kanamycin plates with 20μl and 100μls respectively. </p><br>
 +
 
<p><b><u>Wednesday 1st August</u></b></p>
<p><b><u>Wednesday 1st August</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>Only a few small colonies were found on the plate for BBa_I0500 + BBa_B0034 this morning. </p>
+
<p>Only a few small colonies were found on the plate for BBa_I0500 + BBa_B0034 this morning. </p><br>
 +
 
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
-
<p>•<u>Transformation:</u></p>  
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>Transformation</u></a></p>  
<p>Constitutive promoter BBa_J23119, 2012 Distribution Kit Plate 1, 18A</p>
<p>Constitutive promoter BBa_J23119, 2012 Distribution Kit Plate 1, 18A</p>
<p>Using Invitrogen TOP10 competent cells. </p>
<p>Using Invitrogen TOP10 competent cells. </p>
<p>Spread on two ampicillin plates with 20μl and 100μls respectively. </p><br>
<p>Spread on two ampicillin plates with 20μl and 100μls respectively. </p><br>
-
<p>•<u>Adding cultures</u></p>   
+
 
 +
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Transferring colonies to liquid medium</u></a></p>   
<p>Cells transformed with BBa_I0500 + BBa_B0034 were added into liquid medium and incubated overnight</p>
<p>Cells transformed with BBa_I0500 + BBa_B0034 were added into liquid medium and incubated overnight</p>
<p>Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic. </p><br>
<p>Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic. </p><br>
 +
<p><b><u>Thursday 2nd August</u></b></p>
<p><b><u>Thursday 2nd August</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
<p>Good colonies were found on the 150μl BBa_J23119 plates this morning. They were placed in the fridge ready for later. </p><br>
<p>Good colonies were found on the 150μl BBa_J23119 plates this morning. They were placed in the fridge ready for later. </p><br>
-
<p>•<u>Mini-Prepping</u> of BBa_I0500 + BBa_B0034 by Chris and Sophie the work experience students</p>
+
<p>• <a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Miniprepping</u></a> of BBa_I0500 + BBa_B0034 by Chris and Sophie our work experience students</p>
 +
 
 +
<p>BBa_I0500 + BBa_B0034 was nanodropped and showed no DNA. A gel was run to test this and it confirmed no DNA was present. </p><br>
-
<p>BBa_I0500 + BBa_B0034 was nanodropped and showed no DNA. A gel was run to test this and it was shown that no DNA was present. </p><br>
 
<p>•<u>Gel Electrophoresis</u> was run to check whether any DNA was present and to confirm the nanodrop readings after digestion using the EcoR1 and Pst1 enzymes. The gel was set up and run as 100ng of DNA. A 1% agarose gel was used. </p>
<p>•<u>Gel Electrophoresis</u> was run to check whether any DNA was present and to confirm the nanodrop readings after digestion using the EcoR1 and Pst1 enzymes. The gel was set up and run as 100ng of DNA. A 1% agarose gel was used. </p>
<p>Gel Electrophoresis showed no bands. </p><br>
<p>Gel Electrophoresis showed no bands. </p><br>
 +
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
<p>Streak plates were then made with the remaining broth from the BBa_I0500 + BBa_B0034 culture growth and incubated overnight at 37°C. </p><br>
<p>Streak plates were then made with the remaining broth from the BBa_I0500 + BBa_B0034 culture growth and incubated overnight at 37°C. </p><br>
-
<p>•<u>Adding cultures</u></p>   
+
 
-
<p>Cells transformed with BBa_J23119 were added into liquid medium and incubated overnight</p>
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Transferring colonies to liquid medium</u></a></p>   
 +
<p>Cells transformed with BBa_J23119 were added to liquid medium and incubated overnight.</p>
<p>Protocol was followed using ampicillin for the BioBrick part as the selection antibiotic. </p><br>
<p>Protocol was followed using ampicillin for the BioBrick part as the selection antibiotic. </p><br>
Line 164: Line 179:
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>•<u>Mini-Prepping</u> of BBa_J23119</p>
+
<p>•<a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Miniprepping</u></a> of BBa_J23119</p>
-
<p>The protocol was changed slightly. After the neutralisation solution had been added, the eppendorfs were centrifuged for 10 minutes rather than 5 in order to get a better pellet formation. </p>
+
<p>The protocol was changed slightly. After the neutralisation solution had been added, the eppendorfs were centrifuged for 10 minutes rather than 5 in order to obtain a better pellet formation. </p>
-
<p>40ul of water was added instead of 40µl in order to make sure the concentration of DNA increased. </p>
+
<p>40ul of water was added instead of 50µl in order to make sure the concentration of DNA increased. </p>
<p>BBa_I0500 was nanodropped and recorded good concentrations. </p>  
<p>BBa_I0500 was nanodropped and recorded good concentrations. </p>  
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Latest revision as of 00:05, 27 September 2012

ExiGEM2012 Lab Book 3GP wk4

The 3-Gene Inducible Plasmid: 30th July - 3rd August 2012

Monday 30th July

Afternoon

•Transferring colonies to liquid medium

Cells transformed with BBa_I0500 were added into liquid medium and incubated overnight.

Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic.


Tuesday 31th July

Morning

• Miniprepping

BBa_I0500

BBa_I0500 was nanodropped and recorded poor concentrations.


•Gel Electrophoresis

Run to check fragment sizes of BBa_I0500 after digestion using the EcoR1 and Pst1 enzymes

Gel Electrophoresis showed a faint band at the correct size.


• Sequencing

BBa_K094120_ BBa_B0034 was sent off for sequencing using the primers VF2 and VR.


•3A Assembly

BBa_I0500 + BBa_B0034

Performed with the NEB protocol but using the Linearized Plasmid Protocol from the parts registry. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. As described in the Linearized Plasmid Protocol, a mastermix was made and 4μl of this was added to 4μl of destination plasmid for the digestion.


Afternoon

•Transformation

BBa_I0500 + BBa_B0034

Using Invitrogen TOP10 competent cells.

Spread on two kanamycin plates with 20μl and 100μls respectively.


Wednesday 1st August

Morning

Only a few small colonies were found on the plate for BBa_I0500 + BBa_B0034 this morning.


Afternoon

•Transformation

Constitutive promoter BBa_J23119, 2012 Distribution Kit Plate 1, 18A

Using Invitrogen TOP10 competent cells.

Spread on two ampicillin plates with 20μl and 100μls respectively.


•Transferring colonies to liquid medium

Cells transformed with BBa_I0500 + BBa_B0034 were added into liquid medium and incubated overnight

Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic.


Thursday 2nd August

Morning

Good colonies were found on the 150μl BBa_J23119 plates this morning. They were placed in the fridge ready for later.


• Miniprepping of BBa_I0500 + BBa_B0034 by Chris and Sophie our work experience students

BBa_I0500 + BBa_B0034 was nanodropped and showed no DNA. A gel was run to test this and it confirmed no DNA was present.


•Gel Electrophoresis was run to check whether any DNA was present and to confirm the nanodrop readings after digestion using the EcoR1 and Pst1 enzymes. The gel was set up and run as 100ng of DNA. A 1% agarose gel was used.

Gel Electrophoresis showed no bands.


Afternoon

Streak plates were then made with the remaining broth from the BBa_I0500 + BBa_B0034 culture growth and incubated overnight at 37°C.


•Transferring colonies to liquid medium

Cells transformed with BBa_J23119 were added to liquid medium and incubated overnight.

Protocol was followed using ampicillin for the BioBrick part as the selection antibiotic.


Friday 3rd August

Morning

•Miniprepping of BBa_J23119

The protocol was changed slightly. After the neutralisation solution had been added, the eppendorfs were centrifuged for 10 minutes rather than 5 in order to obtain a better pellet formation.

40ul of water was added instead of 50µl in order to make sure the concentration of DNA increased.

BBa_I0500 was nanodropped and recorded good concentrations.

The promoter was too small to show up on a 1% gel electrophoresis.

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