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Team:Groningen/Labwork/27 June 2013
From 2013.igem.org
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| - | <p>Because of the low concentrations a new PCR is done using the PCR products. | + | <p>Because of the low concentrations a new <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> is done using the PCR products. |
<p>A <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> is done for some different silk constructs to start with. The following combinations are made: | <p>A <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> is done for some different silk constructs to start with. The following combinations are made: | ||
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<br>For every sample an annealing temperature of 50°C is used, expecting a size of 900 bp. | <br>For every sample an annealing temperature of 50°C is used, expecting a size of 900 bp. | ||
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| + | <p>The samples are run over a 0.8% <a href="https://2013.igem.org/Team:Groningen/protocols/GelElectrophoresis"><FONT COLOR="black"><b>agarose gel</b></FONT></a>. This revealed that no bands are present at all. So the gel is stained with a high concentration of Ethidium Bromide and the bands appear. A big smear is seen for all the silk products, with a higher concentrated band around 200 bp. Because of this results it is decided to do a gradient PCR on the first combination from 55-75°C. | ||
<p> | <p> | ||
Latest revision as of 17:01, 28 July 2013
Mirjam
Added 1 ul serva/100ml to the earlier made 0.8% agarose gel.Today the PCR products of the signal sequences (FliZ, EstA, MotB and LytB) are purified using the Roche gel purification kit. Using nanodrop reveals the following concentrations:
| signal sequence | concentration (ng/µl) |
|---|---|
| FliZ | 9.2 |
| LytB | 8.2 |
| MotB | 8.8 |
| EstA | 8.8 |
Because of the low concentrations a new PCR is done using the PCR products.
A PCR is done for some different silk constructs to start with. The following combinations are made:
- signal sequence - N-terminal strep tag - silk
- signal sequence - silk - C-terminal strep tag
- signal sequence - silk - no strep tag
- silk without tag
For every sample an annealing temperature of 50°C is used, expecting a size of 900 bp.
The samples are run over a 0.8% agarose gel. This revealed that no bands are present at all. So the gel is stained with a high concentration of Ethidium Bromide and the bands appear. A big smear is seen for all the silk products, with a higher concentrated band around 200 bp. Because of this results it is decided to do a gradient PCR on the first combination from 55-75°C.
Claudio
The following bibliography summarizes the source of inspiration on which the heat-attracted bacteria sub-project is based on:- Thermal Regulation of Membrane Lipid Fluidity by a Two-Component System in Bacillus subtilis by L. M. Bredeston, D. Marciano, D. Albanesi, D. De Mendoza, and J. M. Delfino
- The three adaptation systems of Bacillus subtilis chemotaxis by Christopher V. Rao, George D. Glekas and George W. Ordal
- Chemotaxis in Bacillus Sutilis: how bacteria monitor environmental signals by Liam F. Garrity and George W. Ordal
- Regulation of Bacillus subtilis DesK thermosensor by lipids by Mariana Martin and Diego De Mendoza
