EPF-Lausanne/21 July 2009

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===Wet Lab===
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<font size="6" color="#007CBC"><i>21 July 2009</i></font>
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----
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==Wet Lab==
The transformation of the Term plasmids worked, so we did glycerol stocks of the 4 new Term and the death cassette.
The transformation of the Term plasmids worked, so we did glycerol stocks of the 4 new Term and the death cassette.
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As the ligation this way seems not to work (either the enzymes for the digestion aren't working, either there is another problem in the ligation), we decided to try another way to do it : by doing a 2 step PCR. We designed and ordered the primers, now we just have to wait for them to go on in our cloning of LovTAP. Have a look at the cloning strategy to see how we designed the primers.
As the ligation this way seems not to work (either the enzymes for the digestion aren't working, either there is another problem in the ligation), we decided to try another way to do it : by doing a 2 step PCR. We designed and ordered the primers, now we just have to wait for them to go on in our cloning of LovTAP. Have a look at the cloning strategy to see how we designed the primers.
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The idea of 2 step PCR : [[Media:2step pcr‎.pdf]]
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The idea of [[Media:2step pcr‎.pdf|two step PCR]]
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==Cloning Strategy==
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===Cloning Strategy===
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One useful website to know the restriction sites of enzymes:  
One useful website to know the restriction sites of enzymes:  
<br>http://www.genscript.com/cgi-bin/products/enzyme.cgi?op=all_ez
<br>http://www.genscript.com/cgi-bin/products/enzyme.cgi?op=all_ez
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the first step introduces the LacI promoter and the RBS upstream the LovTAP gene with the Forward primer, whereas the Reverse introduces the Term downstream.
the first step introduces the LacI promoter and the RBS upstream the LovTAP gene with the Forward primer, whereas the Reverse introduces the Term downstream.
In the second step we only introduce the E-X restriction sites upstream and the SP downstream.
In the second step we only introduce the E-X restriction sites upstream and the SP downstream.
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==People in the lab==
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: Nathalie, Caroline, Mélanie, Tu, Heidi
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Latest revision as of 08:58, 28 July 2009



Contents

21 July 2009



Wet Lab

The transformation of the Term plasmids worked, so we did glycerol stocks of the 4 new Term and the death cassette.

Then miniprep of the 5 transformation products.

Digestion following the iGEM protocol : we cut with X and P Term, E and P the backbone and we take a LovTAP that was digested Friday with E and S.

Running of a gel to check the digestion products. Actually it seems the digestion didn't work. We did a second gel, by loading more product, and we clearly got the same result.

As the ligation this way seems not to work (either the enzymes for the digestion aren't working, either there is another problem in the ligation), we decided to try another way to do it : by doing a 2 step PCR. We designed and ordered the primers, now we just have to wait for them to go on in our cloning of LovTAP. Have a look at the cloning strategy to see how we designed the primers.

The idea of two step PCR

Cloning Strategy

One useful website to know the restriction sites of enzymes:
http://www.genscript.com/cgi-bin/products/enzyme.cgi?op=all_ez

The restriction site used were:
EcoRI GAATTC
XbaI TCTAGA
SpeI ACTAGT
PsiI TTATAA

Design the primers for the 2 step-PCR: the first step introduces the LacI promoter and the RBS upstream the LovTAP gene with the Forward primer, whereas the Reverse introduces the Term downstream. In the second step we only introduce the E-X restriction sites upstream and the SP downstream.


People in the lab

Nathalie, Caroline, Mélanie, Tu, Heidi



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