Obtain larger dna fragments after digestion and gel cleaning - (Sep/09/2005 )
I have been trying to clone a gene fragment of 1000kb into pCDNA3.
After running the PCR (with a temp gradient in order to be the most selective) I obtain a distinct band of the right size. When I gel clean and digest with hindIII and xhoI I obtain extra bands of 2000 and 4000kb.
I ve tried running a mock gel clean to see if that was contaminated, but it was clear, I ve also ran some loading dye on its own and it wasnt that.
I also ran the gel cleaned product before any digestion, and found that the bigger bands already appear here.
It seems that my insert is ligating itself as the band sizes are multiples of its initial size.
Does anybody have an idea? Is there maybe a way to prevent the insert of binding to itself?
(I have gel cleaned the insert, done many ligations and transformed, but I do not get any colonies).
Thanks for any help
Did you stain it with EtBr before gel extraction?
It is not suggested to use EtBr for large fragment like 1000kb.
Crystal violet is better with this case.
Thanks for your reply. I usually stain the gel with EtBr and it has been working well for fragments of 1030kb. I have also made 2 chimeras of the gene I am now having problems cloning, using overlap PCR and EtBr in the gel. I dont think the problem comes from the gel or staining. It looks more like the gene is self ligating in some way.
Thanks for the help... i need it!!