Team:University College London/LabBook/Week14

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'''Method:''' http://2012.igem.org/Team:University_College_London/Protocols/Competency2
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<html><div class="protocol protocol-Generic">Bla bla bla</div><div class="protocolContent"></html>{{:http://2012.igem.org/Team:University_College_London/Protocols/Competency2}}<html></div></html>
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http://2012.igem.org/Team:University_College_London/Protocols/Competency2
== Tuesday 11.09.12 ==  
== Tuesday 11.09.12 ==  

Revision as of 00:56, 27 September 2012

Contents

Monday 10.09.12

Aim: Day 2 of Generating competent cells. The aim is to incubate cells in the presence of CaCl2 to prepare the cell wall, allowing it to become permeable to DNA for transformation.


Method:


Bla bla bla


http://2012.igem.org/Team:University_College_London/Protocols/Competency2

Tuesday 11.09.12

Aim: Day 3 of Generating competent cells


Method: http://2012.igem.org/Team:University_College_London/Protocols/Competency3

Wednesday 12.09.12

Aim: To carry out a transformation using the 3A ligation products in order to be able to analyse whether ligation was successful.


Method: http://2012.igem.org/Team:University_College_London/Protocols/Transformation1


Thursday 13.09.12

Aim:


Picking of colonies from the ligation transformation plates


Methods: http://2012.igem.org/Team:University_College_London/Protocols/ColonyDNAExtraction#DNA_Extraction_from_Colonies


Friday 14.09.12

Aim:


To check whether the 3A have carried out the ligation successfully by purifying the O/N culture from the previous day, carrying out an analytical digest and analysing results on a gel.


Methods:


A mini-prep was carried out as per the following protocol:


http://2012.igem.org/Team:University_College_London/Protocols/Miniprep2


This was followed by a 10ul analytical digest as per the following:


http://2012.igem.org/Team:University_College_London/Protocols/EnzDig2


Finally, results of the figest were run on a gel as follows:


http://2012.igem.org/Team:University_College_London/Protocols/Electrophoresis


Results:


The following digram shows results of the analytical digest, with each purified DNA being cut once and twice in order to confirm identity:

BioBricks BioBrick Size (bp) Ligation size (bp) Plasmid Backbone Plasmid Expected Size (bp) Combined Size
BBa_K729001 & BBA_J23100 933 & 35 968 PSB1C3 2070 3038
BBa_K729001 & BBa_J23119 & BBa_B0034 933 & 35 & 12 980 PSB1C3 2070 3050
BBa_K729002 & BBa_J23119 & BBa_B0034 484 & 35 & 12 531 PSB1C3 2070 2601


Conclusion:


The bands on the gel indicate are not as expected. Hence, it is concluded that the ligation was unsuccessful.



Tuesday 11.09.12

Step 1 - Aim:


To generate enough plasmid backbone (PSB1C3) to be used in a 3A ligation. This is followed by a PCR clean-up.


Methods:


http://2012.igem.org/Team:University_College_London/LabBook/Protocols/PCR#Transformation_Protocol_2


Step 2 - Aim:


To check whether PCR is successful. After the PCR reaction, we can detect the presence of the correct products by running a 1% gel electrophoresis.


Methods:


http://2012.igem.org/Team:University_College_London/Protocols/Electrophoresis


Results:


The following gel results indicate whether PCR was successful.


UCL2012Week14B.png


Conclusion:

Since band sizes were as expected, the PCR was considered to be successful. Hence, the backbone was purified and used in the 3A ligation.


Thursday 13.09.12

Aim:

To generate enough linker (BBa_J23119 + BBa_B0034) to be used in a 3A ligation. This is followed by a PCR clean-up.


Methods: http://2012.igem.org/Team:University_College_London/LabBook/Protocols/PCR#Transformation_Protocol_2


Step 2 - Aim:

To check whether PCR is successful. After the PCR reaction, we can detect the presence of the correct products by running a 1% gel electrophoresis.


Methods:


http://2012.igem.org/Team:University_College_London/Protocols/Electrophoresis


Results:


The following gel results indicate whether PCR was successful.


UCL2012Week14A.png


Conclusion:


Since we obtained the expected bands in the gel, the PCR was considered successful and the purified DNA used in the 3A ligation.


Friday 14.09.13

Aim: To characterise nuclease activity BL21 cells


Method: To add protocol


Results:


The following table shows readings of the diameters and OD at different time points:


Date Time Colony diameter/mm Halo diameter/mm Absorbance at 600 OD
Friday 12.30 0 0 0
Friday 16.30 0 0 0
Friday 19.30 0 0 0
Friday 22.30 0 0 0
Saturday 01.30 0.5 1.5 0.068
Saturday 04.30 1 2 0.098
Saturday 07.30 1.5 3 0.159
Saturday 10.30 1.5 3.5 0.192
Saturday 12.30pm 2 4 0.203
Saturday 14.30 2 4 0.209
Saturday 16.30 2.5 5 0.215


The average depth of the colonies was noted to be 0.5 - 1mm


Conclusion:


It can be seen that nuclease works as expected in BL21 cells. However, nuclease activity is less efficient when compared to nuclease activity in WnU cells. This is in line with expectations, since nuclease is found naturally in WnU cells but not in BL21 cells.