Team:Groningen/Labwork/24 July 2013

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'''Mirjam'''
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<br/>Run the samples of the PCR made 23-07 over a 1.5% agarose gel. The samples show the presence of the expected bands for 3 out of 4 signal sequences. So it is tried to obtain these samples by Gel extraction.
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Made a restriction digestion of the rest of the samples of silk 2 and 3 (strepF-R and F-strepR) to obtain a higher concentration.
 
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The primers that are ordered for heat motility are found and diluted. A PCR is made to obtain Pdes, CheY RBS.
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'''Inne'''
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<h2>Mirjam</h2>
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<br/>Run the samples of the PCR made 23-07 over a 1.5% agarose <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>gel</b></FONT></a>. The samples show the presence of the expected bands for 3 out of 4 signal sequences. So it is tried to obtain these samples by Gel extraction. No bands are seen on gel.
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Made a <a href="https://2013.igem.org/Team:Groningen/protocols/Digestion"><FONT COLOR="black"><b>restriction digestion</b></FONT></a> of the rest of the samples of silk 2 and 3 (strepF-R and F-strepR) to obtain a higher concentration. With the restriction digest a <a href="https://2013.igem.org/Team:Groningen/protocols/Ligation"><FONT COLOR="black"><b>ligation</b></FONT></a> to the four signal sequences is made.
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The primers that are ordered for heat motility are found and diluted. A <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> is made to obtain Pdes, CheY RBS, the samples are present. So a PCR purification is done.
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New <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR's</b></FONT></a> are made for all three silks and the four signal sequences.
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<h2>Inne</h2>
<br>Made a -80 stock of the Munich BBa_k823023 backbone + our IPTG promoter.
<br>Made a -80 stock of the Munich BBa_k823023 backbone + our IPTG promoter.
<br>Made a miniprep of the inoculated cells with the k077557 biobrick that Chicago requested.
<br>Made a miniprep of the inoculated cells with the k077557 biobrick that Chicago requested.
<br>Concentration of the final sample is 21.1 ng/uL.
<br>Concentration of the final sample is 21.1 ng/uL.
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<br>Sample was stored in -20 freezer.
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<br>Sample was stored in -20 freezer
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<br>Determined concentration of PCR samples pdes and cheY by nanodrop. pdes 151.2 ng/uL and cheY 71.4 ng/uL.
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<br>Ran a 1.5% <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>gel</b></FONT></a> with both samples, at 90 V for 24 minutes. well 1:marker, well 2:cheY, well 3:pdes.
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<h2>Claudio</h2>
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<br>The plates are taken out from the incubator and the presence of RFP is detected using UV light.</br>
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<img src="https://2013.igem.org/File:Munich_woIPTG.jpg" alt="Plate1" width="40%">
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<br><b>PICUTRES</b></br>
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Latest revision as of 11:52, 30 July 2013

Mirjam


Run the samples of the PCR made 23-07 over a 1.5% agarose gel. The samples show the presence of the expected bands for 3 out of 4 signal sequences. So it is tried to obtain these samples by Gel extraction. No bands are seen on gel. Made a restriction digestion of the rest of the samples of silk 2 and 3 (strepF-R and F-strepR) to obtain a higher concentration. With the restriction digest a ligation to the four signal sequences is made. The primers that are ordered for heat motility are found and diluted. A PCR is made to obtain Pdes, CheY RBS, the samples are present. So a PCR purification is done. New PCR's are made for all three silks and the four signal sequences.

Inne


Made a -80 stock of the Munich BBa_k823023 backbone + our IPTG promoter.
Made a miniprep of the inoculated cells with the k077557 biobrick that Chicago requested.
Concentration of the final sample is 21.1 ng/uL.
Sample was stored in -20 freezer

Determined concentration of PCR samples pdes and cheY by nanodrop. pdes 151.2 ng/uL and cheY 71.4 ng/uL.
Ran a 1.5% gel with both samples, at 90 V for 24 minutes. well 1:marker, well 2:cheY, well 3:pdes.

Claudio


The plates are taken out from the incubator and the presence of RFP is detected using UV light.
Plate1
PICUTRES

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