EPF-Lausanne/8 October 2009

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==Wet Lab==
==Wet Lab==
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===Colony PCR===
 
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On the same double transformants -> 2 min extension.
 
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===Gel===
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Since the Trp-mutated strains which contained only one plasmid still managed to grow in our media even though there was a double antibiotic selection, we wondered whether they contained an antibiotic resistance which we didn't know of. To verifiy this, we put all three strains in culture either in LB+ampicilin or in LB+kanamycin. In theory only one of the strains (JW 4356-2) should grow, in the medium with kanamycin. The other 2 strains (JRG 465 and LRG 1046) shouldn't be able to grow at all. We will check tomorrow morning.
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Here the 2nd strains seems to have a double transformant. But we let the gel overnight so there is no BrEth any more, we had to take a time of exposure of 38.2 s to see something. We will do a nicer gel this afternoon.
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===Preparation of the time-course experiment===
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The idea is to look at the evolution of fluorescence over the time, for different time of exposition on the light.
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7 ml of LB + 1 ml of DH5a RO2.4 + BB1 #3 overnight culture.
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5 conditions :
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- + light + IPTG - Trp
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- + light - IPTG - Trp
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- - light + IPTG - Trp
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- - light - IPTG + Trp
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- - light - IPTG + Trp
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- - light - IPTG - Trp
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IPTG at 1 mM (100 mM sol. diluted 1/100). Trp diluted 1/25. Incubated in conditions at 37°C. Measures will be taken with plate reader every 30 minutes starting at  1h.
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===Miniprep===
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We did a miniprep of new strain double transformants to confirm gel results by digestion assay :
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- RO1.1 + BB1 JRG 1046 (n°1,3,7)
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- RO2.4 + BB1 JRG 1046 (n°1,3,6)
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+ miniprep of DH5 a RO2.4 + BB1 #3 (strain that works) to have the 2 plasmids for subsequent transformations.
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===Digestion assay===
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For each clones, we use digestion by P and S (since LovTAP contains 2 P sites and Trp op contains 2 S sites). For reaction, incubate at 37°C (INCUB37). Digestion of about 1h30.
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===Gel===
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Once again to see the double transformants + of digestion assay.
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Result ?? TO BE COMPLETED
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==People in the lab==
==People in the lab==
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Heidi, Gab, Tu
 
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Basile, Tu, Gab
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Latest revision as of 10:12, 10 October 2009



Contents

8 October 2009




Wet Lab

Since the Trp-mutated strains which contained only one plasmid still managed to grow in our media even though there was a double antibiotic selection, we wondered whether they contained an antibiotic resistance which we didn't know of. To verifiy this, we put all three strains in culture either in LB+ampicilin or in LB+kanamycin. In theory only one of the strains (JW 4356-2) should grow, in the medium with kanamycin. The other 2 strains (JRG 465 and LRG 1046) shouldn't be able to grow at all. We will check tomorrow morning.

People in the lab

Basile, Tu, Gab


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