Problem during Electroporation - in vivo electroporation (Mar/26/2008 )
I digested one DNA by BamHI and EcoRI and both are dephosphorylated.then I digest another DNA by XhoI and SacII and both the end is dephosphorylated and digest the vector with EcoRI and SacII then by using ligation I got the linear DNA at the correct size area. When I apply this linear DNA to the cell by electroporation 2.5kV and 4.9ms I am not able to get the plasmid containing the inserted DNA. The isolated colony contains only the vector. No insert there.
the 3` and 5` ends were also checked. And there is also similarities between two fragment.
Can anyone suggest me ???
It is not completely clear to me what you mean, but it seems that you are dephosphorylating your inserts, when you should be dephosphorylating the vector, so that it can't religate. So, my suggestion is to dephosphorylate your vector (but not the insert) and try to do a ligation with vector+insert and another one with just vector (this will show if your dephosphorylation worked).