Proceeding with partial digests may work, but it is going to be trick, since you have already three EcoRI sites in your plasmid (one in the vector and two in the dimeric insert). You may try to optimize the EcoRI digestion by using different digestion times or enzyme concentrations and then gel purify the desired fragment, but yields will likely not be very good. I would suggest that you try to PCR out your fragment using primers that hybridize to the vector and that are designed to flank the insert with adequate restriction sites. One side is easy since it already contains an XbaI site. For the other side, you could either add the restriction site at the 5' end, or, if it is critical to excise the insert with no further addition, you could design a primer that overlaps the junction between vector and insert and introduces a restriction site at the desired place. Make sure that the 3' segment overlapping the insert is no longer than 10-12 bp to prevent amplification of the monomer lying downstream. Gel purify the fragment of the desired length and recut it with the appropriate enzymes. As a matter of precaution, resequence your fragment after cloning, but if you use a high fidelity polymerase for the amplification, mutations occur very rarely.

Good luck

Pierre

You havent given us sufficient information. Is the dimer head to head, head to tail, etc, and what orientation in relation to the next vector (which we might guess is an exprssion vector??). And does the insert contain the entire CDS (assuming you are looking to express an encoded protein)?

Thanks for the response @Pierre; I would try the partial digest; I also wanna ask per adventure I saw the expected band of interest without the others, can i extract it and proceed with cleaning? could it mean that EcoRI cuts once in the vector (which is what i want anyway).

@ Geoff, the dimer is head to tail, It is not an expression vector; I will be cloning this dimer insert in an expression vector. Thanks for your time.

I see.

I think the partial EcoRI will work, and under the right conditions you will have some product that is cut only at the insert site that is furthest from the XbaI cut. That should be easy to see on a gel versus the doubly digested insert bands. But that assumes that the XbaI site is at the 5' (amino-terminal coding) end of the insert(s) and that your expression vector is 5' XbaI - 3' EcoRI and that the insert is cloned in frame or provides the only initiation codon downstream of the Shine-Dalgarno seq in the vector. Etc.

But achieving partials is tricky and my experience is that you need far less restriction endonuclease than you think! I would carry out a pilot digest with 1:10 or 1:3 serial dilutions of EcoRI for 1 hour at 37oC then run gels.

To avoid all this, cant you try cloning again but using much less insert relative to vector (I always try to achieve as equimolar amounts)?

if you have one EcoRI site inside your insert. Then you would have two EcoRI sites in your dimer and cutting with EcoR1 with destroy the message whether you perform a partial digest or complete digest and you would not get a complete dimer excised from the vector. It would be good if you could upload a diagram of your cloned fragment with Restriction sites shown.

Re partial digest: I agree with Geoff about the pilot before setting up the preparative digest. If you manage to get the fragment with the proper size, you can gel purify it and reclone it. But be sure to start the digest with enough material (several µg). Because, as Matthew's gospel has it :

"For unto every one that hath shall be given, and he shall have abundance: but from him that hath not shall be taken away even that which he hath."