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ligation problem - (Oct/20/2004 )


I want to insert a 500bp into a 4k vector. After running the ligation rxn, I do the transformation and isolate the plasmid from the cells. Now what I want to know is that whether it is the desire ligated plasmid.
So I try digesting the isolated plasmid using XmnI which will cut the supposed ligated plasmid twich and give two linear DNAs with one 2K and another 3K. After incubation for 16h at 37 degree,I run the gel to see what is going on.
The gel shows 3 bands: one around 2k but faint,one around 3k very strong,and one around 5k also very faint. In the lane of the uncut plasmid,I saw only one band with length around 2k. It is really unreasonable since it goes so quickly and where is the nicked?
I am really confused. Is there any way I can quickly check whether the ligation is successful or not? or anyone has ever met such a question?
Any suggestion will be greatly appreciated!



Well first the two expected band shouldn't be 2k and 3k, as your ligated plasimd has a total length of only 4500 bp. I suppose these were only rough estimations? Anyway it basically looks like your restriction was not complete.

To quickly check your clones i recommend to make Eckhardt gels (or quick lysis) of your clones with plasmids (does your plasmid allow X-gal selection?) and as marker put in the empty vector (ideally also add a plasmid with the anticipated size, 4.5 kb in this case). select for those that are larger than the empty vector and only isolate them. You are aware that CCC plasmids are much faster than linear DNA?

With these plasmids make your restrictions (ideall two to be very sure). Ideally these should cut in insert and vector resulting in different bands depending on the direction of insertion.
16h is awfully long for restriction. depending on the units i use i hardly restrict longer than 2-3 hours at max. beware of star activity of some enzymes if incubate too long. Other than that check if you used the right buffer and if you used enough enzyme for your DNA (otherwise dilute your plasmid).