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Team:Heidelberg LSL/Notebook-14/03/2012
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| - | Screening of the four different biobrick clonings done by colony-PCR. | + | *<b>Screening of the four different biobrick clonings done by colony-PCR.</b><br/> |
| - | Protocol: | + | <b>Protocol:</b><br/> |
| - | A reaction mix was prepared containing 0.2 | + | A reaction mix was prepared containing 0.2 µl of each screening primer used for the screening, 9.6 µl of water and 10 µl of 2x PCR mastermix (fermentas). Finally a colony was picked from a plate using a sterile pipette tip and was dipped into the PCR mix a few times. 7-8 clones were screened for each different cloning setup as follows:<br/> |
| - | SulA-GFP and SulA-LacZ: primers SulA_fw and VR (standard sequencing primer) were used. Only in case SulA was successfully cloned into the backbone containing the reporter gene, we would get a PCR product. GFP would give a product of roughly 1000 bp, LacZ one of roughly 3500 bp. | + | **<u>SulA-GFP and SulA-LacZ:</u> primers SulA_fw and VR (standard sequencing primer) were used. Only in case SulA was successfully cloned into the backbone containing the reporter gene, we would get a PCR product. GFP would give a product of roughly 1000 bp, LacZ one of roughly 3500 bp.<br/> |
| - | RecA-GFP and RecA-LacZ: primers VF2 and VR (standard sequencing primers) were used. We compared the product size of the different clones to the product size from the original vectors from the registry (only containing recA or LacZ). In case the cloning was successful, we should ssee a 200 bp shift in product size, which can be detected by gel electrophoresis. | + | **<u>RecA-GFP and RecA-LacZ:</u> primers VF2 and VR (standard sequencing primers) were used. We compared the product size of the different clones to the product size from the original vectors from the registry (only containing recA or LacZ). In case the cloning was successful, we should ssee a 200 bp shift in product size, which can be detected by gel electrophoresis.<br/> |
| - | The PCR program was done as follows: | + | The PCR program was done as follows:<br/> |
| - | 94°C/3min||94°C/30s|60°C/30s|72°C/3min 45s||30x 72°C/10min|4°C/forever | + | <p style="font-family:monospace">94°C/3min||94°C/30s|60°C/30s|72°C/3min 45s||30x 72°C/10min|4°C/forever</p> |
Afterwards, 3 ul PCR product were loaded onto a 1 % agarose gel. | Afterwards, 3 ul PCR product were loaded onto a 1 % agarose gel. | ||
Revision as of 12:05, 7 June 2012
Notebook
Welcome to our notebook!
Here you will find the documentation of our laboratory work of the last few month in diary form. This notebook comprises the work in three phases:
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Biosensor Construction - 14/03/2012
- Screening of the four different biobrick clonings done by colony-PCR.
Protocol:
A reaction mix was prepared containing 0.2 µl of each screening primer used for the screening, 9.6 µl of water and 10 µl of 2x PCR mastermix (fermentas). Finally a colony was picked from a plate using a sterile pipette tip and was dipped into the PCR mix a few times. 7-8 clones were screened for each different cloning setup as follows:
- SulA-GFP and SulA-LacZ: primers SulA_fw and VR (standard sequencing primer) were used. Only in case SulA was successfully cloned into the backbone containing the reporter gene, we would get a PCR product. GFP would give a product of roughly 1000 bp, LacZ one of roughly 3500 bp.
- RecA-GFP and RecA-LacZ: primers VF2 and VR (standard sequencing primers) were used. We compared the product size of the different clones to the product size from the original vectors from the registry (only containing recA or LacZ). In case the cloning was successful, we should ssee a 200 bp shift in product size, which can be detected by gel electrophoresis.
- SulA-GFP and SulA-LacZ: primers SulA_fw and VR (standard sequencing primer) were used. Only in case SulA was successfully cloned into the backbone containing the reporter gene, we would get a PCR product. GFP would give a product of roughly 1000 bp, LacZ one of roughly 3500 bp.
The PCR program was done as follows:
94°C/3min||94°C/30s|60°C/30s|72°C/3min 45s||30x 72°C/10min|4°C/forever
Afterwards, 3 ul PCR product were loaded onto a 1 % agarose gel.
