Blue/white screening

Dilute IMMEDIATELY with 40 p, TE Buffer (10 mM Tris, pH 8.0,1 mM EDTA) and either transform IMMEDIATELY or freeze the reaction until you are ready to transform. coli with a transformation efficiency in excess of 1 x 10 are recommended and if the vector has been modified for blue-white screening ensure that an appropriate E. coli host strain is used 5 pi of the diluted reaction should give tens to hundreds of colonies per well of a 24-well plate. 

Pick colonies for plasmid miniprepping as usual (if blue-white screening is used then blue colonies should constitute <10% if the reactions were successful. (The blue colonies are derived from inefficiently linearized parental plasmid and should not be picked as they are non- recombinant ). Two colonies are normally sufficient to find a recombinant clone but more may be required. 

Pol. mini-Tn5 transposon using lacZ blue/white screen, then delivering the transposon to stable insertion in host chromosome. Another mini-Tn5 provides T7 polymerase inducible by benzoate. Seven different antibiotic selections available to allow multiple genes expressed in same host. ... 

Ligated plasmids should be checked for insertion of the desired fragment instead of just religation, most conveniently by blue-white screening. In vectors with the lacZ gene sequence, insertion at any restriction site of the MCS results in disruption of that sequence so that no /i-galactosidase is expressed and the synthetic X-gal substrate cannot form blue colonies. 

Recall What is blue/white screening What is the key feature of a plasmid that is used for it ... 

The key feature of a plasmid capable of blue/white screening is the gene for the a-subunit of the enzyme p-galactosidase. These plasmids are used with a strain of E. coli that are deficient in the a-subunit of this enzyme. Galactosidase can convert a colorless sugar derivative, called X-gal, to a... 

We routinely use ampicillin selection of transformants and blue-white screening for transformants with an insert in the vector. We then confirm that the insert is... 

Blue-white screen F-PCR product is ligated to pGEM-T and transformed... 

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