DNA Purification from gels - (Nov/29/2005 )
I heard that it is possible to get contamination of the "old" vector in the band containing the fragment you want to transfer into other vector. I wonder how is it possible to get a huge molecule like a plasmid in the gel fraction of a tiny cDNA (~500bp). Does anybody heard of this? If so, what would be the explanation? Thanks a lot!!
While I have been known to be wrong at times, I believe this is more of a problem if your insert is similar in size to the vector post digestion. Of course you also have to be careful about where on the gel the blade touches while you cut so as not to touch the vector band either.
This is not a very frequent problem but does happen from time to time you can check the last topics on the forium and I'm sure you'll find some discussions on the subject (I actively participate to one of them some weeks ago)
The explanation is very simple when the starting vector is not entirely cut you can get traces of supercoiled vector which will of course generate more recombinants than your ligation cause SC transformation efficiency is generating 100 times more colonies than ligated products.
The rest is just about overloading the preparative slots for example which leads to the trapping of the SC vector in the insert band !
agree with wath pesji said. Perhaps run a bit of undigested plasmid on a gel together with cut to see the migration.
Thanks for your reply!
I'll do digest controls specific for my plasmid of interest after I'm cloning.
I wonder how oft people do this...never did myself