Team:Heidelberg LSL/Notebook-14/03/2012

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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:

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.
    

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.



Fig. 1 Colony-PCR screen of RecA-GFP construct. No detectable shift of any screened clone can be seen compared to the control. Screen has to be repeated.


Fig. 2 Colony-PCR screen of RecA-LacZ construct. Most clones show a small shift upwards in product length. Furthermore, they show a second band at 1200 bp, probably due to unspecific primer binding.


Fig. 3 Colony-PCR screen of SulA-GFP (#3.1-3.8) and SulA-LacZ (#4.1-4.8). Only clones containing the SulA promoter can give a PCR product of 1000 bp (SulA-GFP) or 3700 bp (SulA-LacZ). Positive clones are i.e. #3.4, #4.2, #4.8.

The screening gave positive clones for all constructs (fig. 2, fig. 3), but not the recA-GFP construct (fig. 1). Therefore this screen has to be repeated using a larger number of colonies.

• 5 ml LB-Amp overnight cultures were inoculated for clones #2.3, #3.4 and #4.8.