Team:SDU-Denmark/labwork/Notebook/week9

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<p> <b>27-08-2012  to  02-09-2012</b> </p>
<p> <b>27-08-2012  to  02-09-2012</b> </p>
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<p>DNA purification from FFT liquid cultures were made.<br/></br>
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<p>
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Nanodrop was performed on it:<br/>
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After 48 hours in the incubator, the 1-FFT colonies finaly reached a high enough concentration for sequencing.<br/><br/>
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1: 83,6 ηg/μL<br/>
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2: 17,2 ηg/μL<br/>
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3: 121,1 ηg/μL<br/>
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4: 48,1 ηg/μL<br/>
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5: 99,1 ηg/μL<br/>
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6: 59,6 ηg/μL<br/>
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7: nothing(an error must have occurred)<br/>
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8: 59,6 ηg/μL<br/></br>
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We made check-digest with EcoRI+SpeI to examine the plasmids in preparation of mutagenesis.
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The check digest showed that some had the appropriate digested sizes we would expect from a plasmid of 3kb and a gene of 2kb.<br/></br>
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Mutagenesis was run on these FFT colonies containing all primers.<br/>
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We made new SST cultures again and plated them on AMP-resistance plates. <br/>
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Isolated FFT and SST plasmids and sent them for sequencing.<br/>
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Afterwards we did a digest and transformation on FFT and plated them.<br/>
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We made liquid cultures of FFT and SST and incubated them over night. Later we made transformation on the three parts we chose from the iGEM kit.<br/>
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1) R0010 <br/>
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2) E0040<br/>
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3) B0015<br/>
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Afterwards we plated them on AMP-resistance plates. <br/>
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We made liquid cultures of the three parts<br/>
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Purification of SST, FFT, B0015, E0040 and R0010 and following PCR
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The O.N. cultures of SST, FFT and our 3 parts (B0015/terminator, E0040/GFP and R0010/LacL) showed promising results of growth. A miniprep was made from the 32 samples and a Nanodrop as well which gave the following results:
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</p>
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<TABLE border="1" width="100%">
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<CAPTION><EM>NanoDrop Results</EM></CAPTION>
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<TR><TH>1. R0010, culture 1 <TD>18,0 ng/ul<TH>2. B0015, culture 3 <TD>67,9 ng/ul
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<TR><TH>3. B0015, , culture 5<TD>62,9 ng/ul<TH>4. R0010, culture 3 <TD>70,5 ng/ul
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</TABLE>
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Meanwhile, all the SST colonies had died out. To avoid unnecesary excess work, we simply ran PCR with all mutagenesis primers on an older 1-SST containing pJET plasmid. Miraculous, both gel band length and check digest on the resulting sequence yielded perfect results. Why had we not done this before?<br/><br/>
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In the following days 1-SST ligated into pJET, grown on ampicillin plates, transformed into Top10, transfered to liquid medium and miniprepped for sequencing.<br/>
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</p>
 
 

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

27-08-2012 to 02-09-2012

After 48 hours in the incubator, the 1-FFT colonies finaly reached a high enough concentration for sequencing.

Meanwhile, all the SST colonies had died out. To avoid unnecesary excess work, we simply ran PCR with all mutagenesis primers on an older 1-SST containing pJET plasmid. Miraculous, both gel band length and check digest on the resulting sequence yielded perfect results. Why had we not done this before?

In the following days 1-SST ligated into pJET, grown on ampicillin plates, transformed into Top10, transfered to liquid medium and miniprepped for sequencing.

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