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Why did I find a Clonation fragment with a molecular weight unexpected? - (Mar/28/2010 )

I amplified a gen by PCR using like template a recombinat plasmid that contained the interest gene. this template is in the vector PET100D-topo; the primers used have a restriccion sites in the 5' terminal for enzymes BamHI and NotI. so the PCR fragment was digested with BamHI and NotI, and and finally it was cloned in an eukaryotic vector non-comercial (pTUBNT), for this reason I dont have vector sequence and I cant design primers: I just could do a colony PCR amplification with gen primers to identify my interest fragment into the E. coli Top10 bacterial colonies previously transformed, I got an specific amplification product but the band has a lower molecular weight compared with positive control. the diference between two fragments is about 20-30 bp. Could someone gives me a reason for this?...


Possibly there was some secondary structure or incomplete sequence replication in the bit you cloned into the new vector.


I think an incomplete replication couldn't be possible because if it were lost some terminal nucleotides of the sequence, The gene couldn't amplify because the primers can't annealing. and regarding the secundary structure posibility, could you explain it more deeply?...
Thanks for your help