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Team:Groningen/Labwork/24 July 2013
From 2013.igem.org
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<h2>Mirjam</h2> | <h2>Mirjam</h2> | ||
| - | <br/> </html>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. | + | <br/> </html>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. |
| - | 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. | + | 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. |
| - | 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. | + | 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. |
| - | New PCR's are made for all three silks and the four signal sequences. | + | 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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<br>Determined concentration of PCR samples pdes and cheY by nanodrop. pdes 151.2 ng/uL and cheY 71.4 ng/uL. | <br>Determined concentration of PCR samples pdes and cheY by nanodrop. pdes 151.2 ng/uL and cheY 71.4 ng/uL. | ||
| - | <br>Ran a 1.5% gel with both samples, at 90 V for 24 minutes. well 1:marker, well 2:cheY, well 3:pdes. | + | <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. |
<h2>Claudio</h2> | <h2>Claudio</h2> | ||
Revision as of 11:50, 30 July 2013
Mirjam
Run the samples of the PCR made 23-07 over a 1.5% agarose <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis">gel</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.
Made a <a href="https://2013.igem.org/Team:Groningen/protocols/Digestion">restriction digestion</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">ligation</a> to the four signal sequences is made.
The primers that are ordered for heat motility are found and diluted. A <a href="https://2013.igem.org/Team:Groningen/protocols/PCR">PCR</a> is made to obtain Pdes, CheY RBS, the samples are present. So a PCR purification is done.
New <a href="https://2013.igem.org/Team:Groningen/protocols/PCR">PCR's</a> 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.
PICUTRES
