ligation rxn on gel was a smear

[dakims]

ligation rxn on gel was a smear

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Post by dakims » Wed Jan 20, 2010 6:32 pm

i ran a ligation reaction on a gel and got a very faint smear. the smear was so faint i may even be wrong. i loaded 5ul from my ligation which had .1ug dna total. I'm really not sure what happened. I ran a control with everything but t4 ligase and i got two bands. so contaminant can't be in the buffer or water or dna sample. so something had to be wrong the the ligase? i dont have undigested vector (i sap'd it all) to run it against so i'm not sure what to do! i went ahead with the transformation praying that it will work. but i think i should run another ligation. any suggestions?!

[JackBean]

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Post by JackBean » Wed Jan 27, 2010 7:49 am

Yeah, you probably will have to try another ligation. Best try some other ligase.

[clevermizo]

Re: ligation rxn on gel was a smear

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Post by clevermizo » Thu Jan 28, 2010 4:03 pm

With that much DNA in there you probably have a lot of incorrect products. Typically I use <100 ng total DNA (i.e., 50 ng vector and 3:1 molar excess of insert). I can't really recommend using 1 ug of DNA in a ligation reaction. Now, if you use as small of an amount of DNA as I do, you won't likely be able to resolve your ligation products easily on a gel (I dunno, maybe you can). But I just transform and screen colonies. Anyway, this seems to work out. This month I had to make 15 constructs, so hopefully I know whereof I speak.

Of course I don't know all your reaction conditions, amounts of vector/insert, units of ligase, reaction time, etc. You mind sharing that?