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Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


Restriction analysis

Restriction analysis - Molecular Cloning Forum

Restriction analysis - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 03-08-2014, 04:43 PM
Pipette Filler
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Default Restriction analysis



Hello,

I would like to get some help with something. I cloned a gen in pET TOPO vector 101 and when a did the analysis of the transformants with restriction enzymes( double digestion) I did not get what I was really expecting in the agarose gel. I just got one plasmid with the right bands that corresponded with the plasmid with the gen in the correct orientation, but the bands were very light. Furthermore, with the other plasmids I got bands that were not the bands I was expecting at all, not even if the gen was not in the correct orientation or the plasmid did not have the gen. I am not sure why I got these bands, I checked more than one time the bands I supposed I was going to get with those enzymes. Also, I am not sure either why the bands are as light. If someone could give some answer it would be very helpful.

Thanks.
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Old 03-08-2014, 04:48 PM
Pipette Filler
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Default Re: Restriction analysis

When I said the bands were light I meant not very bright.
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