Team:SDU-Denmark/labwork/Notebook/week10

From 2012.igem.org

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We made a TAQ PCR on the three parts. The TAQ PCR on the three parts showed about 500 bp for all parts, which is too low. It looks like something is wrong with the primers.
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To bypass the growth problem of our bacteria, we spent this week on preparing to transfer the genes into iGEM plasmids.<br/><br/>
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We also ran a gel on the digested parts.</br>
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Ligation of the amplificated and miniprepped parts:</br>
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E0040(GFP) and B0015(Terminator) was ligated into pSB1K3.(E0040+B0015)</br>
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FFT was ligated into pSB1C3</br>
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SST was ligated into pSB1C3</br>
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All of the ligations were transformed into E. coli TOP10, plated and incubated.
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For characterization we prepared:</br>
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SST - E.coli, pJET 1.2, Ampicillin medium</br>
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FFT - E.coli, pJET 1.2, Ampicillin medium</br>
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Transformed TOP10 with ampicillin resistance, ampicillin medium.</br>
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Not-transformed TOP10 without ampicillin resistance, plain medium without resistance.
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These cultures were made for later characterization of our genes impact on bacterial growth conditions.
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(There was no growth from the SST gene)
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We did a miniprep on RFP+Terminator (J04650)
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This week we also ordered new iGEM primers.
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The plates we incubated earlier had plenty of colonies. Some were chosen and transferred to liquid medium.
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While preparing the plasmids we also prepared cultures of the promoter R0010, GFP coding sequence E0040 and terminator B0015.<br/><br/>
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Colony PCR was made, in the meantime we plated the bacteria where our genes should be with the promoter and made new liquid cultures.
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At the end of the week we attempted digestion and ligation into pSB1C3, but something mysterious and unexpected happened: we got red colonies, and we had no idea where they came from. We hadnt touched any RFP containing sequences and the partsregistry page for the linearized plasmid said nothing of RFP in the biobrick. Our best guess is that the linearized plasmid is isolated from something that contain RFP.
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Unfortunately, colony PCR only showed chromosomal DNA and that GFP + terminator worked.
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Because of this, we made a PCR amplification on all of our genes and ran a gel.
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Due to the continued lack of results we made PCR on ALL our mini-preps to check if they do in fact contain our genes.
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We made PCR on SST and FFT and ran a gel to check the lengths.  
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Latest revision as of 02:15, 27 September 2012

iGEM TEAM ::: SDU-DENMARK

Laboratory Notebook

1st week 2nd week 3rd week 4th week
5th week 6th week 7th week 8th week
9th week 10th week 11th week 12th week

03-09-2012 to 09-09-2012

To bypass the growth problem of our bacteria, we spent this week on preparing to transfer the genes into iGEM plasmids.

While preparing the plasmids we also prepared cultures of the promoter R0010, GFP coding sequence E0040 and terminator B0015.

At the end of the week we attempted digestion and ligation into pSB1C3, but something mysterious and unexpected happened: we got red colonies, and we had no idea where they came from. We hadnt touched any RFP containing sequences and the partsregistry page for the linearized plasmid said nothing of RFP in the biobrick. Our best guess is that the linearized plasmid is isolated from something that contain RFP.

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