DNA degradation while digestion. - (Sep/25/2007 )
hi... i am trying to clone a 130 bp insert in pFF-19 (4.2 kb) vector. im getting recombinant colonies (2-3 colonies only) which give good amplification with insert specific and vector specific primers of required size, so im sure that insert is there in the vector (though less number of colonies do worry me). on isolating plasmid of recombinant colonies and digesting them with BamHI and KpnI, im getting a smear. I have tried chaniging my enzymes, buffers, water, even comp. cells but still problem is there.
im clueless about what is going on! can anybody help me please...
if you change your digestion reagents, i would rather think of a problem in your extraction procedure.
Either you may have genomic DNA which causes a smear, or you vortex too mmuch your plasmid resulting in shearing.
Otherwise you may have star activity during your digestion.
Either you may have genomic DNA which causes a smear, or you vortex too mmuch your plasmid resulting in shearing.
Otherwise you may have star activity during your digestion.
i have checked uncut plasmid on gel, so im sure that there is no genomic dna. as a rule i never vortex my plasmid, and star activity is also taken much into account.. so im still clueless, where is the mistake!
did you try single digestion and séquential digestion ?