HELP! - cloning problem

MSRA MSRA at novo.dk
Thu Nov 5 01:32:20 EST 1998


     Bernard pointed out the following comment.  I'm sorry that my 
     explanation was not enough.  
     
     As to pMAL, it has lacZ and I did blue/white screening on a replica 
     plate - then discarded it.  Because cells bearing a pMAL will 
     eventually die after IPTG induction.  However, I do not think pBAD has 
     lacZ, so I didn't do blue/white screening.
     
     I used primers that should anneal to a plasmid on each side of MCS.  I 
     clean up fragment from a gel with QIAquick.  THe insert fragment 
     looked good on a gel.
     
     Bernard, I appreciate your suggestion.  I will hold on screening more 
     clonies.
     
     Mari
     
     
     ------------------------------------------------------------
     If "pMAL" is NEB's vector for fusion expression I am pretty sure
     it does not contain LacZ so you can't do blue/white screening
     (*all* the colonies should be white). I'm not familiar with
     pBAD but it could be the same situation.
     
     PCR screening is pretty reliable in my hands. Did you have
     a good positive control? Are your primers for the plasmid
     or for the insert? Are you swamping the reaction with
     contaminating empty vector or free fragment? 
     
     
     How did you clean up the fragment? Did it look clean on a gel?
     Did you check your cut vector after digestion (and cleanup)?



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