EPF-Lausanne/8 October 2009

From 2009.igem.org

(Difference between revisions)
(→Wet Lab)
(→Wet Lab)
 
(4 intermediate revisions not shown)
Line 26: Line 26:
==Wet Lab==
==Wet Lab==
-
===Colony PCR===
 
-
On the same double transformants -> 2 min extension.
 
-
===Gel===
+
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.
-
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.
+
-
 
+
-
===Preparation of the time-course experiment===
+
-
The idea is to look at the evolution of fluorescence over the time, for different time of exposition on the light.
+
-
 
+
-
7 ml of LB + 1 ml of DH5a RO2.4 + BB1 #3 overnight culture.
+
-
 
+
-
5 conditions :
+
-
 
+
-
- + light + IPTG - Trp
+
-
 
+
-
- + light - IPTG - Trp
+
-
 
+
-
- - light + IPTG - Trp
+
-
 
+
-
- - light - IPTG + Trp
+
-
 
+
-
- - light - IPTG + Trp
+
-
 
+
-
- - light - IPTG - Trp
+
-
 
+
-
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.
+
-
 
+
-
===Miniprep===
+
-
We did a miniprep of new strain double transformants to confirm gel results by digestion assay :
+
-
 
+
-
- RO1.1 + BB1 JRG 1046 (n°1,3,7)
+
-
 
+
-
- RO2.4 + BB1 JRG 1046 (n°1,3,6)
+
-
 
+
-
+ miniprep of DH5 a RO2.4 + BB1 #3 (strain that works) to have the 2 plasmids for subsequent transformations.
+
-
 
+
-
===Digestion assay===
+
-
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.
+
-
 
+
-
===Gel===
+
-
Once again to see the double transformants + of digestion assay.
+
==People in the lab==
==People in the lab==
-
Heidi, Gab, Tu
 
 +
Basile, Tu, Gab
<html><center><a href="https://2009.igem.org/EPF-Lausanne/7_October_2009"><img src="https://static.igem.org/mediawiki/2009/thumb/6/61/Flèche_gauche.png/70px-Flèche_gauche.png"></a>   
<html><center><a href="https://2009.igem.org/EPF-Lausanne/7_October_2009"><img src="https://static.igem.org/mediawiki/2009/thumb/6/61/Flèche_gauche.png/70px-Flèche_gauche.png"></a>   

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


Retrieved from "http://2009.igem.org/EPF-Lausanne/8_October_2009"