mung bean nuclease

Richard Daines rdaines at q.continuum.net
Sun Dec 10 21:15:43 EST 1995


I'm a little confused...are you religating the mungbean digest and then 
transforming into E. coli?  If you are then the possible outcomes are: a 
blunt end ligation (successful mungbean digesting) or remaining, unblunted 
EcoRI sites coming back together (unsuccessful mungbean digestion).  
Having tried a similar digest on an Nco-I site I can relate.  I screened 
about 40 colonies before finding what I thought was a correct one.  Yes 
the Nco-I site was gone but so was an extra base which created a fatal 
frame shift.  

Is it possible to dephosphorylate after the mungbean and blunt-end ligate 
in an additional piece of DNA.  This will cut down on the background.

If not possible, increase digestion time or reduce the amount of DNA.




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