I have a problem about pIRES2-DsRed2 (5.3kb,clotech) double digestion. I used XhoI and BamHI to digest the pIRES2-DsRed2 and I got two bands, which are both around 2500bp. I should to be only one band. And then I extracted the fragment (5.3kb) from the gel. I ligated this fragment with my insert fragment(700bp, digested with same enzymes from another plasmid). I transformed the bacteria and picked up the colony. I redigested the plasmid again with same enzymes. To my suprise, these two bands (around) appeared again. After that I use same plasmid( pIRES2-DsRed2 )from another lab , I got same phenomenon. I do not know what happened! I am crazy , could you give me some suggestions.
figure 1 lane 1 is the marker ; lane 2 is pIRES2-DsRed2 double digestion with XhoI and BamHI ; lane 3 is another plamid double digestion with XhoI and BamHI . arrow head indicate these bands around 2500bp.
figure2 lane 1 is marker. lane 2-6 is ligated plasmid double digestion with XhoI and BamHI . All of them have my insert fragment.arrow head indicate these bands around 2500bp.
Edited by tantao, 19 July 2010 - 12:28 AM.