I am running a ligation reaction to one of the sticky ends of lambda-DNA.

The oligo for the ligation is 25 bp (10x excess), whereas lambda-DNA is 48 kbp. I would like to recover the lambda-DNA and discard the unreacted excess oligo. Given their vastly different molecular weights, they should be easy to separate, but I could not find a standard methodology online.

I do not want to run gel and my total sample volume is 0.5 mL. There are some filtration columns that may work, but the input volume is only 25 uL (I have 20 times that).

I don't have to get rid of all the oligo, having slightly more lambda-DNA than oligo at the end would suffice.

Which simple protocol may work in this case?


A spin column might work for your purposes since you can do multiple rounds of binding if your volume is too large to bind in a single pass, but for DNA of that size a lot will be irreversibly stuck to the column. See for example the protocol for the Monarch® PCR & DNA Cleanup Kit.

Consequently I would recommend just doing an ethanol precipitation, which favors recovery of longer DNA molecules (see for example this protocol).

  • $\begingroup$ Nice answer. The Monarch stuff only seems to work for DNA up to 25 kpb (may not work for my 48 kbp). My lambda-DNA didn't seem to go trough the gel filtration column, I may take a look into that ethanol precipitation. $\endgroup$ – Brenlla Aug 7 at 10:19

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