I am making genomic DNA library. I fragmented genomic DNA using Sonication and used DNA blunting kit to prepare fragments for ligation. On the other hand, plasmid vector was digested to generate blunt ends using one enzyme (SmaI) and dephosphorylated using CIAP.
Vector: Insert ratio's of 1:1 to 1:5 were tested and analysed on 1% agarose gel. All ratios worked giving bands larger than linearized vector band.
When any of these were used to transform chemical competent (tested with pUC DNA and give good efficiency at all time when transformation is done) and commercial electrocompetent cells, No colonies are found!
Interestingly, when the ligation reaction is re-analysed in 1% agarose gel, the reaction has reversed, linear vector and DNA fragments smear can be seen on the gel. Ligation is repeated with DNA fragments and linear vector, analysed on gel, all worked but after transformation still no colony. Then ligation is reversed. I have even tried cleaning the ligation reaction by phenol/chloroform/IAA with ethanol precipitation, no colonies and ligation reaction reversed.
Have anyone ever experienced this (reversing of ligation)? Or anyone with a suggestion.
Now am known as elecrophoresis "dude" because I run these gels like it my profession