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
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.