Team:SDU-Denmark/labnotes2

From 2010.igem.org

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''Results:'' A gel, containing the PCR product was run, using [http://www.fermentas.de/product_info.php?info=p1110 GeneRuler(TM) DNA Ladder Mix, ready-to-use (#SM0333)] loaded in lane one (from the left:<br><br>
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''Results:'' A gel, containing the PCR product was run, using [http://www.fermentas.de/product_info.php?info=p1110 GeneRuler(TM) DNA Ladder Mix, ready-to-use (#SM0333)] loaded in lane one, lane 2 contains sample run at 56,1 degrees celcius and in lane 3 sample run at 64,5 degrees celcius is found.<br><br>
[[Image:Annealing of the two mutated FlhDC strands (sheila).jpg|300px]]<br>
[[Image:Annealing of the two mutated FlhDC strands (sheila).jpg|300px]]<br>
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We can se very faint bands in lane 2 and 3, at around 1000bp. The band in lane 3 is a little more visible than lane 2. Because we haven't used primers ind this PCR, it is possible that the two mutated strands has a lower possibility of annealing to each other. It s however possible to amplify the few complete FlhDCmut strands in a new PCR, which is what we will do next.<br><br>
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We can se very faint bands in lane 2 and 3, at around 1000bp, which is the right size. The band in lane 3 is a little more visible than lane 2. Because we haven't used primers in this PCR, the pieces will only have been extended once and we need to ad flhDC fw and rv in order to obtain more product. <br><br>
--[[User:Sheila|Sheila]] 09:10, 22 July 2010 (UTC)
--[[User:Sheila|Sheila]] 09:10, 22 July 2010 (UTC)
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''Date:'' July 19th <br><br>
''Date:'' July 19th <br><br>
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]<br><br>
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1]<br><br>
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''Method:'' The experiment was done in two rounds. First, the annealing product, made by Pernille on july 19th was amplified. Then we amplified Pernilles amplification simultaneously as we amplified [https://2010.igem.org/Team:SDU-Denmark/labnotes2#Annealing_of_the_two_mutated_strands_of_FlhDC_.28FlhDCmut.29 Sheilas previous annealing].<br> Primers used: FlhDC fw and FlhDC rev <br> Polymerase used: Pfu<br><br>
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''Method:'' The experiment was done in two rounds. First, the annealing product, made by Pernille on July 19th was amplified. Then we amplified Pernilles amplification simultaneously as we amplified [https://2010.igem.org/Team:SDU-Denmark/labnotes2#Annealing_of_the_two_mutated_strands_of_FlhDC_.28FlhDCmut.29 Sheilas previous annealing].<br> Primers used: FlhDC fw and FlhDC rev <br> Polymerase used: Pfu<br><br>
''Notes:'' <br><br>
''Notes:'' <br><br>
''Results:''<br><br>
''Results:''<br><br>
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''Notes:'' The DNA purified in the last experiment was amplified with PRC using VR og VF2 primers and pfu. the normal pfu program was used for running PCR. The PRC product was loaded on a 2% agarose gel. the brick J13002 (promotor+RBS) are 312bp long wich correspond to the visible band on the gel.<br><br>
''Notes:'' The DNA purified in the last experiment was amplified with PRC using VR og VF2 primers and pfu. the normal pfu program was used for running PCR. The PRC product was loaded on a 2% agarose gel. the brick J13002 (promotor+RBS) are 312bp long wich correspond to the visible band on the gel.<br><br>
[[Image:Team-sdu-denmark-Gelextraktionpromotor+rbs.jpg|300px]]<br><br>
[[Image:Team-sdu-denmark-Gelextraktionpromotor+rbs.jpg|300px]]<br><br>
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The DNA was extracted and afterwards the DNA concentration was measured on Nanodrop. the concentration was found to 13,02bg/uL. The DNA is saved and afterward it needs to be cut with restriction enzymes.
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The DNA was extracted and afterwards the DNA concentration was measured on Nanodrop. the concentration was found to be 13,02ng/uL. The DNA was saved and it was cut with restriction enzymes.
== Group: Photosensor ==
== Group: Photosensor ==
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=== Miniprep of BBa_K081005 in pSB1A2 and BBa_R0011 in pSB1A2 from transformation 20/7 ===
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=== Miniprep of [http://partsregistry.org/Part:BBa_K081005 K081005] in pSB1A2 and [http://partsregistry.org/Part:BBa_R0011 R0011] in pSB1A2 from transformation 20/7 ===
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Start date: 22/07    End date: 22/07<br>
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Start date: July 22nd<br>
''Methods:''  Miniprep, gel electrophoresis and nano drop <br><br>
''Methods:''  Miniprep, gel electrophoresis and nano drop <br><br>
''Protocol:''[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2] <br><br>
''Protocol:''[https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2] <br><br>
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''Analysis:''<br>
''Analysis:''<br>
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Sample 1-4 of BBa_K081005 were pooled and frozen and sample 1-4 of BBa_R0011 were too.<br>
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Sample 1-4 of K081005 were pooled and frozen and sample 1-4 of R0011 were too.<br>
<br>
<br>
=== Gel extraction test (GFX vs. Fermentas) ===
=== Gel extraction test (GFX vs. Fermentas) ===
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Start date: 25/07    End date: 25/07<br>
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Start date: July 25th <br>
''Methods:'' Gel electrophoresis, gel extraction and nano drop <br><br>
''Methods:'' Gel electrophoresis, gel extraction and nano drop <br><br>
''Protocol:''[https://2010.igem.org/Team:SDU-Denmark/protocols#DE1.2 DE1.2][https://2010.igem.org/Team:SDU-Denmark/protocols#DE1.3 DE1.3] <br><br>
''Protocol:''[https://2010.igem.org/Team:SDU-Denmark/protocols#DE1.2 DE1.2][https://2010.igem.org/Team:SDU-Denmark/protocols#DE1.3 DE1.3] <br><br>
''Experiment done by:''  Maria<br><br>
''Experiment done by:''  Maria<br><br>
''Notes:'' <br><br>
''Notes:'' <br><br>
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FlhD/C tq PCR product (5 green) is used in the test.<br>
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FlhD/C taq PCR product (5 green) is used in the test.<br>
PCR product is diluted in H2O to reach a sample concentration below 100ng/uL.<br>
PCR product is diluted in H2O to reach a sample concentration below 100ng/uL.<br>
Nanodrop:<br>
Nanodrop:<br>
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</tr>
</tr>
</table><br>
</table><br>
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A total of 70uL is loaded onto a 2% agarose gel.<br>
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A total of 70 microlitres is loaded onto a 2% agarose gel.<br>
For the GFX gel extraction 350mg of gel was used.<br>
For the GFX gel extraction 350mg of gel was used.<br>
For the Fermentas extraction 460mg of gel was used.<br>
For the Fermentas extraction 460mg of gel was used.<br>
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</table><br><br>
</table><br><br>
''Analysis:''<br>
''Analysis:''<br>
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There is extracted more DNA with the fermentas kit. This may be due to the short centrifugation time in the GFX protocol. Furthermore the GFX kit that was used was old and the buffers may have been contaminated.<br><br>
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More DNA is extracted with the fermentas kit. This may be due to the short centrifugation time in the GFX protocol. Furthermore the GFX kit that was used was old and the buffers may have been contaminated.<br><br>
== Group: Retinal ==
== Group: Retinal ==
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=== Transformation of K081005 in pSB1A2 (constitutive promoter and RBS combined),R0011 in pSB1A2, pSB3C5 w. J04450 and pSB3T5 w. J04450 in Top 10 E.Coli ===
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=== Transformation of [http://partsregistry.org/Part:BBa_K081005 K081005] in pSB1A2 (constitutive promoter and RBS combined),[http://partsregistry.org/Part:BBa_R0011 R0011] in pSB1A2, pSB3C5 w. [http://partsregistry.org/Part:BBa_J04450 J04450] and pSB3T5 w. J04450 in Top 10 E.Coli ===
Start date: 19/07    End date: 20/07<br>
Start date: 19/07    End date: 20/07<br>
''Methods:''  ON culture, making competent cells, transformation <br><br>
''Methods:''  ON culture, making competent cells, transformation <br><br>
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--[[User:Tipi|Tipi]] 08:17, 21 July 2010 (UTC)<br><br>
--[[User:Tipi|Tipi]] 08:17, 21 July 2010 (UTC)<br><br>
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=== Coloni PCR of R0011 in pSB1A2 and K081005 in pSB1A2 ===
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=== Coloni PCR of [http://partsregistry.org/Part:BBa_R0011 R0011] in pSB1A2 and [http://partsregistry.org/Part:BBa_K081005 K081005] in pSB1A2 ===
Start date: 20/07    End date: 20/07<br>
Start date: 20/07    End date: 20/07<br>
''Methods:''  Coloni PCR and gel electrophoresis <br><br>
''Methods:''  Coloni PCR and gel electrophoresis <br><br>

Latest revision as of 19:23, 24 October 2010