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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!!

??? ohmy.gif

-shala-

QUOTE (shala @ Jun 1 2005, 09:21 AM)
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!!

???  ohmy.gif



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.

-dobbiewalton-

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.

-AussieUSA-

[/quote]


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.


[/quote]

i hace extracted my fragment on a 1.5% agarose gel, running for 3hours at 80V and i see just one fragment at 500pb.
after the purification o this fragment, i quantifymy DNA on a agorose gel 1.5%with smart lader running and i have still on fragment.

I will try with a 3% agarose gel.

-shala-