Team:Groningen/Labwork/15 July 2013

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'''Sander'''
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<h2>Sander</h2>
<br/>made MC 10x according to protocol B.Subtilis transformation except instead of adding H2O till it 10ml, 10ml total was added.
<br/>made MC 10x according to protocol B.Subtilis transformation except instead of adding H2O till it 10ml, 10ml total was added.
<br/> made MgSO4 1M solution.
<br/> made MgSO4 1M solution.
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'''Mirjam'''
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<h2>Mirjam</h2>
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<br/> Made PCR reactions to obtain a higher silk concentration, but analysis revealed that the expected bands are not present. An attempt to concentrate the friday samples failed for 3 of the 4 tubes.  
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<br/> Made <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> reactions to obtain a higher silk concentration, but analysis revealed that the expected bands are not present. An attempt to concentrate the friday samples failed for 3 of the 4 tubes.  
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Because the transformation failed again last week. This time a new restriction and ligation is made for the promoter and the backbone. A new protocol for transformation is used.
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Because the transformation failed again last week. This time a new <a href="https://2013.igem.org/Team:Groningen/protocols/Digestion"><FONT COLOR="black"><b>restriction</b></FONT></a> and <a href="https://2013.igem.org/Team:Groningen/protocols/Ligation"><FONT COLOR="black"><b>ligation</b></FONT></a> is made for the promoter and the backbone. A new protocol for transformation is used.
<br/>- tubes are prechilled on ice
<br/>- tubes are prechilled on ice
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<br/>- competent ''E.coli'' cells are defrosted on ice
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<br/>- competent <i>E.coli</i> cells are defrosted on ice
<br/>- 10 ul ligation reaction is added to the competent cells, or 2 ul backbone for the positive control
<br/>- 10 ul ligation reaction is added to the competent cells, or 2 ul backbone for the positive control
<br/>- incubation on ice for 30 min
<br/>- incubation on ice for 30 min
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<br/>- plate on Lb agar + ampicillin
<br/>- plate on Lb agar + ampicillin
<br/>- incubate O/N at 37 degrees Celsius
<br/>- incubate O/N at 37 degrees Celsius
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Positive control: backbone munchen 2012, negative control: no dna, double negative control: no DNa and no competent E.coli
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<h2>Inne</h2>
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<br>For heat motility:
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<br>Requested CheY null Bacillus subtilis strain OIB055 from the University of Illinois.
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<br>Received confirmation that strain OIB055 will be shipped to us.
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<br>
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<br>Requested des null Bacillus subtilis strain AKP4 from the Universidad de Beunos Aires.
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<h2>Claudio and Mike</h2>
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<br>The synthetic genes design has been started.
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<br>The idea is to disassemble the silk gene MaSp2 into the smallest subunits (inspired by iGEM Utah 2012) and codon optimize them.
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<br>The process starts: inspired by Brooks the smallest subunits are defined.
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Latest revision as of 10:21, 30 July 2013

Sander


made MC 10x according to protocol B.Subtilis transformation except instead of adding H2O till it 10ml, 10ml total was added.
made MgSO4 1M solution.

Mirjam


Made PCR reactions to obtain a higher silk concentration, but analysis revealed that the expected bands are not present. An attempt to concentrate the friday samples failed for 3 of the 4 tubes. Because the transformation failed again last week. This time a new restriction and ligation is made for the promoter and the backbone. A new protocol for transformation is used.
- tubes are prechilled on ice
- competent E.coli cells are defrosted on ice
- 10 ul ligation reaction is added to the competent cells, or 2 ul backbone for the positive control
- incubation on ice for 30 min
- heat shock on 42 degrees Celsius for 1 min
- incubation on ice for 5 min
- add 1 ml of LB medium
- incubation at 37 degrees Celsius for 1 hour
- centrifugation and resuspend the pellet in 200 ul.
- plate on Lb agar + ampicillin
- incubate O/N at 37 degrees Celsius Positive control: backbone munchen 2012, negative control: no dna, double negative control: no DNa and no competent E.coli

Inne


For heat motility:
Requested CheY null Bacillus subtilis strain OIB055 from the University of Illinois.
Received confirmation that strain OIB055 will be shipped to us.

Requested des null Bacillus subtilis strain AKP4 from the Universidad de Beunos Aires.

Claudio and Mike


The synthetic genes design has been started.
The idea is to disassemble the silk gene MaSp2 into the smallest subunits (inspired by iGEM Utah 2012) and codon optimize them.
The process starts: inspired by Brooks the smallest subunits are defined.

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