differents results in PCR and sequencing!! - (Jun/01/2005 )

I have extracted a fragment of 500pb of a RAPD profil.
I have cloned it into a vector and extract the plasmidic DNA of 6 clones. after a digestion with EcoRI, I have two bands: the first at 3900pb was the plasmid and my insert was not the same for my 6 clones!!!!
two clones have insert a fragment of 500 and the others have insert approximatively fragment of 485 and 510pb.
I sequence 3 of them and blast them: it's three differents fragments.
on my RAPD profile, i had only one fragment at 500pb. I had control my first extraction product and i have only one fragment at 500pb!!

???

I have extracted a fragment of 500pb of a RAPD profil.
I have cloned it into a vector and extract the plasmidic DNA of 6 clones. after a digestion with EcoRI, I have two bands: the first at 3900pb was the plasmid and  my insert was not the same for my 6 clones!!!!
two clones have insert a fragment of 500 and the others have insert approximatively fragment of 485 and 510pb.
I sequence 3 of them and blast them: it's three differents fragments.
on my RAPD profile, i had only one fragment at 500pb. I had control my first extraction product and i have only one fragment at 500pb!!

??? 

Do not be surprised to find multiple "bands' from your RAPD profile and on agarose, it will be very hard to distinguish bands from 485-510 base pairs. Try running your original sample on a precast agarose (like the e-gels from Invitrogen = very thin) or use PAGE. This will allow you to distinguish the different bands and to isolate them.

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Correct if im wrong. It seems you extracted your fragment from an agarose gel? Running for longer period of time at a lower voltage and allowing your pcr to migrate further might give you enough separation to distinguish the three fragment from each other.