User:J. M. Uriel Urquiza/Notebook/pRK404 & pK18Mobsacb standardization/2011/03/14

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(pK404 standardization)
Current revision (14:11, 14 March 2011) (view source)
(pK404 standardization)
 
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==pK404 standardization==
==pK404 standardization==
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I made 5 min partial digestion of pR404 using EcoRI to linearize the vector. EcorI is part of the RFC 10
+
I made a 5 min partial digestion of pRK404 using EcoRI to linearize the vector. EcoRI site is part of the RFC 10
-
so it must be eliminated from pRK404 in orther to be able to use this vector with biobricks.  
+
so it must be eliminated from pRK404 to be able to use this vector with biobricks.  
-
What I am going to do next is fill the 3' overhang and ligate then transform the ligation and select for
+
What I am going to do next is fill the 3' overhang and ligate the vector, then transform the ligation and select for
-
colonies that maintain its blue color in presence of X-gal. By this way i am selecting does plasmids
+
colonies that maintain its blue color in presence of X-gal. By this way I am selecting those plasmids
-
that loose the EcoRI sites that is ta pRK404 origin but maintain de EcoRI site that belongs to lacZ.
+
that loose the EcoRI sites which is at pRK404 origin but maintain de EcoRI site that belongs to lacZ.
   
   
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Propagation of base vector with ccdB using Db3.1

The colonies that were stored at 12ºC where restrained in LB Cb and start to incubate them at 30ºC for overnight

pK404 standardization

I made a 5 min partial digestion of pRK404 using EcoRI to linearize the vector. EcoRI site is part of the RFC 10 so it must be eliminated from pRK404 to be able to use this vector with biobricks.

What I am going to do next is fill the 3' overhang and ligate the vector, then transform the ligation and select for colonies that maintain its blue color in presence of X-gal. By this way I am selecting those plasmids that loose the EcoRI sites which is at pRK404 origin but maintain de EcoRI site that belongs to lacZ.


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