Team:Groningen/Labwork/18 July 2013

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'''Sander'''
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<br/>prepared 20 tubes for use in -80 c Glyserol stock
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<h2>Mirjam</h2>
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A check of the RFP, YFP and GFP plates revealed that GFP did not grow on the plate. RFP and YFP colonies are inoculated in liquid LB medium with cm.
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<br/>Also the new made transformation of the promoter in the backbone showed colonies.
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A mini prep is done for RFP and YFP and the samples are stored in the freezer.
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A <a href="https://2013.igem.org/Team:Groningen/protocols/PCR"><FONT COLOR="black"><b>PCR</b></FONT></a> is made for all silk parts. Only the silk without strep tags showed a band at the expected height. Two samples are purified and checked on gel. A band around 900 bp is seen. Because of the low initial concentration, the samples are combined and concentrated to end up with a concentration of 19 ng/ul. This is a high enough concentration to make a restriction digestion.
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Made an inoculation of the promoter in backbone in liquid LB medium with ampicillin and IPTG. This to see if the colonies are again turning red as the wild type srain does.
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<h2>Sander</h2>
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<br/>prepared 20 tubes for use in -80 c Glyserol stock.
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<br/>created a <a href="https://2013.igem.org/Team:Groningen/protocols/Digestion"><FONT COLOR="black"><b>restriction digestion</b></FONT></a> reaction for silk.
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<table border="1">
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<tr>
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<td>Sample</td>
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<td>componant</td>
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<td>volume (ul) </td>
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</tr>
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<tr>
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<td>silk1</td>
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<td>silk</td>
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<td>5</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI buffer</td>
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<td>2</td>
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</tr>
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<tr>
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<td></td>
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<td>BamHI enzyme</td>
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<td>0,5</td>
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</tr>
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<tr>
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<td></td>
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<td>BSA</td>
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<td>0,5</td>
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</tr>
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</table>
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<br/>MilliQ was added to reach a 20 ul volume.
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<br/> placed in  incubater at 37,2 C at 17:10
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<h2>Inne & Mirjam</h2>
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<br>Cells with BBa_k818000, inoculated overnight, were successfully grown.
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<br>Both negative controls were clear, and the rest of the samples was turbid.
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<br>Did a miniprep of the samples 1 & 2 (the ones with Amp).
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<table border="1">
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<tr>
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<td>#</td>
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<td>composition</td>
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<td>Bacteria</td>
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</tr>
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<tr>
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<td>1</td>
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<td>4mL LB 8uL Amp</td>
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<td>+</td>
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</tr>
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<tr>
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<td>2</td>
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<td>4mL LB 8uL Amp</td>
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<td>+</td>
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</tr>
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<tr>
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<td>3</td>
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<td>4mL LB 8uL Amp (negative control)</td>
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<td>-</td>
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</tr>
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<tr>
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<td>4</td>
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<td>4mL LB</td>
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<td>+</td>
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</tr>
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<tr>
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<td>5</td>
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<td>4mL LB (negative control)</td>
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<td>-</td>
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</tr>
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</table>
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<br>Afterwards concentration of DNA was determined via nanodrop.
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<br>Results: sample 1 = 60.1 ng/uL, sample 2 = 31.8 ng/uL
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<br>During the miniprep the filtered LB medium was poured back into the culture (so cells could grow again).
 +
<br>A -80 C stock was made with 1 mL culture (sample 1). (stored in tray: iGEM 2012 A, compartment 70)
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<h2>Claudio and Mike</h2>
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<br>Claudio and Mike are still working on the design. The focus is to check that all the parts (restriction sites, Strp-tag, RBS) necessary for molecular cloning are in place and added to the synthetic genes sequences.
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<div id="footer">
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  <p>iGEM 2013 Groningen</p>
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Latest revision as of 11:20, 30 July 2013

Mirjam

A check of the RFP, YFP and GFP plates revealed that GFP did not grow on the plate. RFP and YFP colonies are inoculated in liquid LB medium with cm.
Also the new made transformation of the promoter in the backbone showed colonies. A mini prep is done for RFP and YFP and the samples are stored in the freezer. A PCR is made for all silk parts. Only the silk without strep tags showed a band at the expected height. Two samples are purified and checked on gel. A band around 900 bp is seen. Because of the low initial concentration, the samples are combined and concentrated to end up with a concentration of 19 ng/ul. This is a high enough concentration to make a restriction digestion. Made an inoculation of the promoter in backbone in liquid LB medium with ampicillin and IPTG. This to see if the colonies are again turning red as the wild type srain does.

Sander


prepared 20 tubes for use in -80 c Glyserol stock.
created a restriction digestion reaction for silk.
Sample componant volume (ul)
silk1 silk 5
BamHI buffer 2
BamHI enzyme 0,5
BSA 0,5

MilliQ was added to reach a 20 ul volume.
placed in incubater at 37,2 C at 17:10

Inne & Mirjam


Cells with BBa_k818000, inoculated overnight, were successfully grown.
Both negative controls were clear, and the rest of the samples was turbid.
Did a miniprep of the samples 1 & 2 (the ones with Amp).
# composition Bacteria
1 4mL LB 8uL Amp +
2 4mL LB 8uL Amp +
3 4mL LB 8uL Amp (negative control) -
4 4mL LB +
5 4mL LB (negative control) -

Afterwards concentration of DNA was determined via nanodrop.
Results: sample 1 = 60.1 ng/uL, sample 2 = 31.8 ng/uL
During the miniprep the filtered LB medium was poured back into the culture (so cells could grow again).
A -80 C stock was made with 1 mL culture (sample 1). (stored in tray: iGEM 2012 A, compartment 70)

Claudio and Mike


Claudio and Mike are still working on the design. The focus is to check that all the parts (restriction sites, Strp-tag, RBS) necessary for molecular cloning are in place and added to the synthetic genes sequences.

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