Team:AUC TURKEY/Results/Cloning

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<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
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line-height:115%;font-family:"Calibri","sans-serif"'>CLONING THE TOP10 STRAINS</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
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line-height:115%;font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
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line-height:115%;font-family:"Calibri","sans-serif"'>TRANSFORMATION</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Plasmids
 +
containing the BBa_K1291071 (Horseradish peroxidase composite part) and
 +
BBa_K1291299 (Lignin Peroxidase composite part) parts were transferred into our
 +
T10 strains, which were prepared through the competent cell preparation
 +
protocol, using transformation protocols. Colonies were seen in agar plates
 +
that had the appropriate antibiotics after a 16 hour long incubation process. </span></p>
 +
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
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line-height:115%;font-family:"Calibri","sans-serif"'>PLASMID ISOLATION</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'> The
 +
colonies taken from plates were incubated in LB Broth containing AMP for 16
 +
hours. DNA isolation was done on the bacteria culture to check the efficiency.
 +
The adequacy of the amount of the nucleic acid concentration for digestion was
 +
met in the acquired samples and it was seen that the samples had peaks at OD
 +
260, which was captured by the nanodrop.</span><span style='position:relative;
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  <td align=left valign=top><img width=281 height=295
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  src="https://static.igem.org/mediawiki/2014hs/a/a8/Cimage001.jpg"></td>
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  <td></td>
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  <td align=left valign=top><img width=290 height=295
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  src="https://static.igem.org/mediawiki/2014hs/e/e0/Cimage002.jpg"></td>
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 +
</p>
 +
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<p class=MsoNormal style='text-align:justify'><span style='position:relative;
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height=486 src="https://static.igem.org/mediawiki/2014hs/f/f4/Cimage003.jpg"></span><br
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</p>
 +
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<p class=MsoNormal style='text-align:justify'><img width=300 height=372
 +
src="https://static.igem.org/mediawiki/2014hs/9/98/Cimage004.jpg" align=left hspace=12
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vspace=12></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
 +
line-height:115%;font-family:"Calibri","sans-serif"'>Digestion and DNA
 +
Electrophoresis</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Gel
 +
electrophoresis was conducted on the samples so that the enzymes which were
 +
digested with the EcoRI and PstI could be verified in terms of their
 +
wavelength. The acquired results showed that the plasmid transfer process was
 +
successful for the Top10 strains while the NEB10 strains, which were taken from
 +
the stock, were disfunctional thus were unusable.</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
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line-height:115%;font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
 +
line-height:115%;font-family:"Calibri","sans-serif"'>CLONING THE BL-21 STRAINS</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Since
 +
we require a high concentration of proteins in our project, we decided to use the
 +
BL-21 strain of E.coli instead of Top10 strains due to its enhanced protein
 +
synthesis capacity. Plasmids containing the BBa_K1291071 (Horseradish
 +
peroxidase composite part) and BBa_K1291299 (Lignin Peroxidase composite part)
 +
parts were transferred into our BL-21 strains, which were prepared through the
 +
competent cell preparation protocol, using the transformation protocol. The
 +
colonies successfully acquired after a 16-hour long incubation process.</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>PLASMID
 +
ISOLATION</span><span style='position:relative;z-index:251662336;left:-20px;
 +
top:0px;width:624px;height:309px'>
 +
 +
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  <td width=281></td>
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<tr>
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  <td height=307></td>
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  <td rowspan=2 align=left valign=top><img width=309 height=309
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  src="https://static.igem.org/mediawiki/2014hs/1/1b/Cimage005.jpg"></td>
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  <td></td>
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  <td align=left valign=top><img width=281 height=307
 +
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</span><br clear=ALL>
 +
</p>
 +
 +
<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>The
 +
chosen colonies were incubated for 16-hours in AMP containing LB Broth. DNA
 +
Isolation was done to test the efficiency of the transformation. A peek at OD
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260 was observed and sufficient levels of DNA concentration was acquired to
 +
continue to digestion.</span><span style='position:relative;z-index:251664384;
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left:-78px;top:0px;width:757px;height:483px'><img width=757 height=483
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src="https://static.igem.org/mediawiki/2014hs/5/55/Cimage007.gif"></span><br clear=ALL>
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
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<p class=MsoNormal style='text-align:justify'><img width=340 height=357
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<p class=MsoNormal style='text-align:justify'><span style='font-size:16.0pt;
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line-height:115%;font-family:"Calibri","sans-serif"'>Digestion and DNA
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Electrophoresis</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>&nbsp;</span></p>
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<p class=MsoNormal style='text-align:justify'><span style='font-family:"Calibri","sans-serif"'>Gel
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electrophoresis was conducted on the samples so that the enzymes which were
 +
digested with the EcoRI and PstI could be verified in terms of their
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wavelength. The acquired results showed that the plasmid transfer process was
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successful for BL-21 Strains and it was possible to continue to the next step.</span></p>
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Revision as of 03:06, 21 June 2014

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CLONING THE TOP10 STRAINS

 

TRANSFORMATION

 

Plasmids containing the BBa_K1291071 (Horseradish peroxidase composite part) and BBa_K1291299 (Lignin Peroxidase composite part) parts were transferred into our T10 strains, which were prepared through the competent cell preparation protocol, using transformation protocols. Colonies were seen in agar plates that had the appropriate antibiotics after a 16 hour long incubation process.

 

 

PLASMID ISOLATION

 The colonies taken from plates were incubated in LB Broth containing AMP for 16 hours. DNA isolation was done on the bacteria culture to check the efficiency. The adequacy of the amount of the nucleic acid concentration for digestion was met in the acquired samples and it was seen that the samples had peaks at OD 260, which was captured by the nanodrop.



 

 

Digestion and DNA Electrophoresis

 

Gel electrophoresis was conducted on the samples so that the enzymes which were digested with the EcoRI and PstI could be verified in terms of their wavelength. The acquired results showed that the plasmid transfer process was successful for the Top10 strains while the NEB10 strains, which were taken from the stock, were disfunctional thus were unusable.

 

 

 

 

CLONING THE BL-21 STRAINS

 

Since we require a high concentration of proteins in our project, we decided to use the BL-21 strain of E.coli instead of Top10 strains due to its enhanced protein synthesis capacity. Plasmids containing the BBa_K1291071 (Horseradish peroxidase composite part) and BBa_K1291299 (Lignin Peroxidase composite part) parts were transferred into our BL-21 strains, which were prepared through the competent cell preparation protocol, using the transformation protocol. The colonies successfully acquired after a 16-hour long incubation process.

 

 

 

PLASMID ISOLATION


The chosen colonies were incubated for 16-hours in AMP containing LB Broth. DNA Isolation was done to test the efficiency of the transformation. A peek at OD 260 was observed and sufficient levels of DNA concentration was acquired to continue to digestion.

 

Digestion and DNA Electrophoresis

 

Gel electrophoresis was conducted on the samples so that the enzymes which were digested with the EcoRI and PstI could be verified in terms of their wavelength. The acquired results showed that the plasmid transfer process was successful for BL-21 Strains and it was possible to continue to the next step.

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