problem with plasmid digestion - (Jan/16/2013 )
Hi every one,
I have a question about digestion: I transformed my plasmid to Ecoli then purified it, then added teh restriction enzyme(sac I takara) for one restriction site and didnt get a good result (just supercoiled without restriction with lower weight than linear plasmid), so I tried with sacI biolabs and got a sharp 3kb band uppr than former expriment but my band should be 4200 b appx.If my incubation time was low and my plasmid was digested incompletely why didnt see 2 band include ccc and linear form? and if it was complete why the band wasnt upper 4000?need help
How much DNA you are taking and how much enzyme you are adding? (for 1ug we add 10 units of NEB enzyme) Have you purified the plasmid only? Its not the contamination? Is your enzyme working fine? In the restriction digestion, if your plasmid is correct then you should see one linearized band of plasmid if you are not getting it then you need to ask and answer the questions I mentioned.
I added 5ul (0.05unit)of enzyme according to manufacturer and 45 ul DNA (10ng/ul)20 ul buffer , up to 200 with DW, incubated in 37 for 4h. My supervisor believes that I should change my enzyme to new one .I dont know if my plasmid was contaminated ,how can I be sure?(so what is this sharp band upper than standard supercil and lower than the target band?
Generally if plasmid is contaminated , you get multiple bands, but thats not the case with you as you said you are getting single band. Since this band is not the one that you are expecting that is why I said it could be something else, some junk DNA. May be you can check your DNA with some other enzyme, if that gives you the expected bands then the problem is with enzyme but not with DNA.
I tried with a new enzyme and have got a good result,
Thanks for your help.
oh Great!! Most welcome ..and all the best for next step