Hello,

For my genetics class, I had to subclone
LUX ABE into pUC18 in order to make E. coli glow in the dark with the addition
of aldehyde. Unfortunately, I had star activity with the digestions, so it made
it almost impossible to identify the LUX ABE band on my electrophoresis gel.

 

My question is: if an enzyme produces
cohesive ends in optimal conditions, will it continue to produce cohesive ends
in star activity conditions? Or, will it turn to blunt ends?

 

Thank you,

 

Julien

 

Whether a fragment is sticky or blunt basically depends on how the respective strands are nicked. In most cases star activity changes the sites only by a little (they still tend to be palindromic to some extent, for example) chances are that they will produce same ends as they did under normal circumstances. Due to the loss of specificity the overhang sequence may very well be different, however.

Thank you very much!