Isolation restr. fragments from agarose gel by Qiagen kit - cloning fungal genomic DNA (Nov/02/2004 )
I have a big problem with isolation restriction DNA fragments from agarose gel. I am using QIaquick gel extraction kit. Genomic DNA were digested by restriction endonuclease, purificated by fenol/chloroform and ethanol precipitation, loaded into agarose gel (0,8%).The bands about 3,5kbp were excised and gel slice was used. DNA was eluted with ddH2O (30ul) and used for electrophorese (5ul). However,in the gel I saw a smear DNA with two bands: 3,5 kbp and more intenzive 1,5 kbp (whyyyyy????). I need the 3,5 kbp fragment for cloning. When I was trying these fragments ligate into vector and than I used for transformation competent cells E.coli, I´d got only 1 bacterial colony with recombinant plasmid against of number colony of transformants with a empty plasmids. Could be DNA fragments izolated from agarose gel damaged? what could be related to lose of sticky end?
The electrophorese was running in unsterile conditions. It is sterility important during excising agarose gel slice? Can anybody help me?
Sorry for my english..
i dont think there is possibility of contamination but it is certainly possible that DNA to degrade while gel band handling. iT HAS HAPPEN TO ME TOO. TRY BE CAREFUL WHEN YOU EXTRACT THE DNA FROM THE GELL BAN