Undigestable/unrestrictable Plasmid? - (Jan/07/2011 )

Hi All!

I have isolated a megaplasmid of ~365kb from E. coli (transformed in DH10B). For some reason, no restriction enzyme seems to restrict it! I am performing restriction analysis on several plasmids (all between 48.5-145.5) at the same time, and they all restrict. I am therefore presuming it is not something technical. This megaplasmid was, however, restricted by S1 nuclease, otherwise I would not have known its size! I used EcoRV, DraI, KpnI, ApaI, AseI and more. When I tried HaeIII, I did get a smear, but I do not know if this was due to restriction or degradation and it was the only enzyme giving a smear.

Does anyone have any suggestions?

Thanks!

Dear Daphne,

how did you purifed your megaplasmid? The protocols i know use very harsh conditions using phenol/chloroform? ...maybe your preparation is contaminated with residues of chemicals used for purification and therefore prevents the enzymes from working properly? As far as i know S1 nuclease cuts ssDNA ...maybe your plasmid is single stranded?

Regards,
p