Team:University College London/LabBook/Week14

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==14-3==
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==Tuesday 11.09.12==
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'''Step 1 - Aim:'''
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To generate enough plasmid backbone (PSB1C3) to be used in a 3A ligation. This is followed by a PCR clean-up.
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'''Methods:'''
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https://2012.igem.org/Team:University_College_London/LabBook/Protocols/PCR#Transformation_Protocol_2
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'''Step 2 - Aim:'''
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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.
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'''Methods:'''
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https://2012.igem.org/Team:University_College_London/Protocols/Electrophoresis
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'''Results:'''
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The following gel results indicate whether PCR was successful.
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[[File:UCL2012Week14B.png]]
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'''Conclusion:'''
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Since band sizes were as expected, the PCR was considered to be successful. Hence, the backbone was purified and used in the 3A ligation.
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== Thursday 13.09.12 ==
== Thursday 13.09.12 ==

Revision as of 00:39, 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, such that it becomes permeable to DNA


Method: https://2012.igem.org/Team:University_College_London/Protocols/Competency2

Tuesday 11.09.12

Aim: Day 3 of Generating competent cells


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

14-2

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:


https://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:


https://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: https://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:


https://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.