Team:Exeter/lab book/3gip/wk3

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         </p>
         </p>
         <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>
 +
        <p>
 +
        -
 +
        </p>
 +
        <a href="https://2012.igem.org/Team:Exeter/Results/inducible"; style="color:#e30614" target="_blank"><font size="3"><b>Results</b></font></a>
 +
</font>
       </font>
       </font>
     </div>
     </div>
     <!--End Project Division Week Hyperlinks-->
     <!--End Project Division Week Hyperlinks-->
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     </td>
     </td>
    
    
   <td width="850" height="250">
   <td width="850" height="250">
   <!------------INSERT WEEKLY IMAGE HERE------------>
   <!------------INSERT WEEKLY IMAGE HERE------------>
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     <img src="" alt="" title="" width="850" height="250">
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     <img src="https://static.igem.org/mediawiki/2012/c/c0/Exe2012labbookandpic.jpg" alt="" title="" width="850" height="250">
   </td>
   </td>
   </tr>
   </tr>
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<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>•<u> Preparation of IDTE buffer</u></p>
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/11" style="color:#57b947"><u>Preparation of IDTE buffer</u></a></p>
-
Protocol was followed exactly.
+
<p>Protocol was followed exactly. </p><br>
<p><b><u>Tuesday 24th July</u></b></p>
<p><b><u>Tuesday 24th July</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<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>   
-
BBa_K206001 + BBa_B0034
+
<p>BBa_K206001 + BBa_B0034</p>
-
BBa_K206000 + BBa_B0034
+
<p>BBa_K206000 + BBa_B0034</p>
-
BBa_K094120 + BBa_B0034.
+
<p>BBa_K094120 + BBa_B0034. </p>
-
All the constructs were ligated into pSB1C3 using the New England Biolab protocol. The protocol required 500ng of plasmid DNA. Using Linearized Plasmid Backbone this at 25ng/μl this used more plasmid than we hoped.
+
All the constructs were ligated into pSB1C3 using the New England Biolab <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#1d1d1b"><u>Protocol</u></a>. The protocol required 500ng of plasmid DNA. Using Linearized Plasmid Backbone this at 25ng/μl this used more plasmid than we hoped. </p><br>
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
-
• IDT re-suspension and transformation of WbnJ and WfcA
+
<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/11" style="color:#57b947"><u>IDT resuspension</u></a> and <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>transformation</u></a> of <i>wbnJ</i> and <i>wfcA</i></p>
-
Alex and Becca finished the transformation of the WbnJ and WfcA genes while Mary and I continued with the Biobrick transformation.
+
<p>Alex and Becca finished the transformation of the <i>wbnJ</i> and <i>wfcA</i> genes while Mary and I continued with the BioBrick transformation. </p>
-
2ul of DNA was used for each transformation into Invitrogen TOP10 competent cells using the protocol provided.
+
<p>2ul of DNA was used for each transformation into Invitrogen TOP10 competent cells using the protocol provided. </p><br>
   
   
<p><b><u>Tuesday 25th July</u></b></p>
<p><b><u>Tuesday 25th July</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
The 3A assembly and subsequent transformation performed on Tuesday 24th July showed no colonies in the morning. It was thought the problem may be that the BBa_B0034 sequence was too small (12bp). It was decided to run the 3A assembly again and this time alongside the standard assembly to compare.
+
<p>The 3A assembly and subsequent transformation performed on Tuesday 24th July showed no colonies in the morning. It was thought the problem may be that the BBa_B0034 sequence was too small (12bp). We decided to run the 3A assembly again but this time alongside the standard assembly allowing us to compare. </p><br>
-
<p>•<u> 3A assembly</u></p>   
+
 
-
The 3A assembly was repeated for:  
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A Assembly</u></a></p>   
-
BBa_K206000 + BBa_B0034 into pSB1T3
+
<p>The 3A assembly was repeated for: </p>
-
BBa_K094120 + BBa_B0034 into pSB1T3
+
<p>BBa_K206000 + BBa_B0034 into pSB1T3</p>
 +
<p>BBa_K094120 + BBa_B0034 into pSB1T3</p><br>
<p>•<u> Standard assembly</u></p>   
<p>•<u> Standard assembly</u></p>   
-
Standard assembly using BBa_B0034 in the destination plasmid. The amount of DNA used increased to 2000ng and the total H20 was changed from 50μl to 35μl to fit the gel.
+
<p>Standard assembly using BBa_B0034 in the destination plasmid. The amount of DNA used was increased to 2000ng and the total H20 was changed from 50μl to 35μl to fit the gel. </p>
-
For the standard assembly the samples were digested for 1 hour at 37C.
+
<p>For the standard assembly the samples were digested for 1 hour at 37°C.
-
A 50μl 8% gel, using low melting agarose, was prepared.
+
<p>A 50μl 8% gel, using low melting agarose, was prepared. </p>
-
While the gel was drying we used a Quiagen Purification Kit to purify the downstream BBa_B0034 digestion. This was performed using the Qiagen Gel Extraction Protocol exactly.  
+
<p>While the gel was drying we used a Qiagen Purification Kit to purify the downstream BBa_B0034 digestion. This was performed using the Qiagen Gel Extraction Protocol exactly. </p><br>
 +
 
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
<p>•<u> Standard assembly Gel Purification</u></p>   
<p>•<u> Standard assembly Gel Purification</u></p>   
-
The digested upstream DNA, BBa_K206000 and BBa_K094120, were run on the 8% gel. The gel was run for 20 minutes until the dye had run 2 thirds of the length of the gel.
+
<p>The digested upstream DNA, BBa_K206000 and BBa_K094120, were run on the 8% gel. The gel was run for 20 minutes until the dye had run 2 thirds the length of the gel.</p>
-
The gel was taken into the Darkroom, visors were worn and lab coats tucked into gloves. The UV tray pulled out and turned to low. BBa_K206000 and BBa_K094120 showed faint bands but after attempting to cut them out, it was decided that not enough DNA was present for gel purification.
+
<p>The gel was taken into the darkroom, visors were worn and lab coats tucked into gloves. The UV tray was pulled out and turned to low. BBa_K206000 and BBa_K094120 showed faint bands but after attempting to cut them out, it was decided that not enough DNA was present for gel purification. </p><br>
-
<p>•<u> Transformation:</u></p>   
+
 
-
Products of the 3A assembly:
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>Transformation</u></a></p>   
-
BBa_K206000 + BBa_B0034  
+
<p>Products of the 3A assembly: </p>
-
BBa_K094120 + BBa_B0034.
+
<p>BBa_K206000 + BBa_B0034</p>
-
Using Invitrogen TOP10 competent cells split into 2 eppendorfs containing 25μl each.
+
<p>BBa_K094120 + BBa_B0034</p>
-
Spread on two tetracycline plates for each transformation using 20μl and 100μls respectively.  
+
<p>Using Invitrogen TOP10 competent cells split between 2 eppendorfs so that each contained 25μl</p>
 +
<p>Spread on two tetracycline plates for each transformation using 20μl and 100μls respectively. </p><br>
<p><b><u>Wednesday 26th July</u></b></p>
<p><b><u>Wednesday 26th July</u></b></p>
<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>   
-
Pbad promoter BBa_I0500, 2012 Distribution Kit Plate 1, 14N
+
<p>Pbad promoter BBa_I0500, 2012 Distribution Kit Plate 1, 14N</p><br>
-
<p>•<u> Adding cultures</u></p>   
+
 
-
Cells containing BBa_K206000 + BBa_B0034 and BBa_K094120 + BBa_B0034 both showed colonies and were added into liquid medium and incubated overnight
+
<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>   
-
Protocol was followed using Kanamycin for all BioBrick parts as the selection antibiotic.
+
<p>Cells containing BBa_K206000 + BBa_B0034 and BBa_K094120 + BBa_B0034 both showed colonies and were added into liquid medium and incubated overnight</p>
 +
<p>Protocol was followed using kanamycin for all BioBrick parts as the selection antibiotic. </p><br>
 +
 
<p><b><u>Thursday 27th July</u></b></p>
<p><b><u>Thursday 27th July</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
Pbad promoter BBa_I0500 showed colonies and was stored in the fridge until Monday.
+
<p>Pbad promoter BBa_I0500 showed colonies and was stored in the fridge until Monday. </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>   
-
BBa_K206000_BBa_B0034
+
<p>BBa_K206000_BBa_B0034</p>
-
BBa_K094120_ BBa_B0034
+
<p>BBa_K094120_ BBa_B0034</p>
-
Both BBa_K206000_BBa_B0034 and BBa_K094120_ BBa_B0034
+
<p>Both BBa_K206000_BBa_B0034 and BBa_K094120_ BBa_B0034
-
were nanodropped and recorded good concentrations.  
+
were nanodropped and recorded good concentrations. </p><br>
<p>•<u>Gel Electrophoresis</u></p>
<p>•<u>Gel Electrophoresis</u></p>
-
Run to check fragment sizes of BBa_K206000_BBa_B0034 and BBa_K094120_ BBa_B0034 after digestion using the ECOR1 and Pst1 enzymes
+
<p>Run to check fragment sizes of BBa_K206000_BBa_B0034 and <p>BBa_K094120_ BBa_B0034 after digestion using the EcoR1 and Pst1 enzymes</p>
-
Gel Electrophoresis showed no bands but this may have been down to the small size of the inserts.
+
<p>Gel Electrophoresis showed no bands but this may have been down to the small size of the inserts. </p>
-
Streak plates made from the overnight cultures and left on the bench until Monday.
+
<p>Streak plates were made from the overnight cultures and left on the bench until Monday. </p>
   
   
      
      
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<tr align="center">
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 +
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 +
    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
 +
    <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a>  &nbsp;&nbsp;|&nbsp;&nbsp;
 +
    <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p>
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Latest revision as of 00:04, 27 September 2012

ExiGEM2012 Lab Book 3GP wk3

The 3-Gene Inducible Plasmid: 23rd - 27th July 2012

Monday 23rd July

Morning

Preparation of IDTE buffer

Protocol was followed exactly.


Tuesday 24th July

Morning

3A Assembly

BBa_K206001 + BBa_B0034

BBa_K206000 + BBa_B0034

BBa_K094120 + BBa_B0034.

All the constructs were ligated into pSB1C3 using the New England Biolab Protocol. The protocol required 500ng of plasmid DNA. Using Linearized Plasmid Backbone this at 25ng/μl this used more plasmid than we hoped.


Afternoon

IDT resuspension and transformation of wbnJ and wfcA

Alex and Becca finished the transformation of the wbnJ and wfcA genes while Mary and I continued with the BioBrick transformation.

2ul of DNA was used for each transformation into Invitrogen TOP10 competent cells using the protocol provided.


Tuesday 25th July

Morning

The 3A assembly and subsequent transformation performed on Tuesday 24th July showed no colonies in the morning. It was thought the problem may be that the BBa_B0034 sequence was too small (12bp). We decided to run the 3A assembly again but this time alongside the standard assembly allowing us to compare.


3A Assembly

The 3A assembly was repeated for:

BBa_K206000 + BBa_B0034 into pSB1T3

BBa_K094120 + BBa_B0034 into pSB1T3


Standard assembly

Standard assembly using BBa_B0034 in the destination plasmid. The amount of DNA used was increased to 2000ng and the total H20 was changed from 50μl to 35μl to fit the gel.

For the standard assembly the samples were digested for 1 hour at 37°C.

A 50μl 8% gel, using low melting agarose, was prepared.

While the gel was drying we used a Qiagen Purification Kit to purify the downstream BBa_B0034 digestion. This was performed using the Qiagen Gel Extraction Protocol exactly.


Afternoon

Standard assembly Gel Purification

The digested upstream DNA, BBa_K206000 and BBa_K094120, were run on the 8% gel. The gel was run for 20 minutes until the dye had run 2 thirds the length of the gel.

The gel was taken into the darkroom, visors were worn and lab coats tucked into gloves. The UV tray was pulled out and turned to low. BBa_K206000 and BBa_K094120 showed faint bands but after attempting to cut them out, it was decided that not enough DNA was present for gel purification.


Transformation

Products of the 3A assembly:

BBa_K206000 + BBa_B0034

BBa_K094120 + BBa_B0034

Using Invitrogen TOP10 competent cells split between 2 eppendorfs so that each contained 25μl

Spread on two tetracycline plates for each transformation using 20μl and 100μls respectively.


Wednesday 26th July

Afternoon

Transformation

Pbad promoter BBa_I0500, 2012 Distribution Kit Plate 1, 14N


Transferring colonies to liquid medium

Cells containing BBa_K206000 + BBa_B0034 and BBa_K094120 + BBa_B0034 both showed colonies and were added into liquid medium and incubated overnight

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


Thursday 27th July

Morning

Pbad promoter BBa_I0500 showed colonies and was stored in the fridge until Monday.

Miniprepping

BBa_K206000_BBa_B0034

BBa_K094120_ BBa_B0034

Both BBa_K206000_BBa_B0034 and BBa_K094120_ BBa_B0034 were nanodropped and recorded good concentrations.


Gel Electrophoresis

Run to check fragment sizes of BBa_K206000_BBa_B0034 and

BBa_K094120_ BBa_B0034 after digestion using the EcoR1 and Pst1 enzymes

Gel Electrophoresis showed no bands but this may have been down to the small size of the inserts.

Streak plates were made from the overnight cultures and left on the bench until Monday.

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