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Why do I have a low efficiency of recombination into my destination vector with my ORFeome Collaboration clone?

The usual way to verify whether an entry clone is working is to run a parallel reaction with the same destination vector and another control entry clone that you have verified to work well. All the ORFeome Collaboration (OC) entry clones are supposed to have been fully sequence verified, including sequence through the proximal ~20 nts 5' and 3' to the insert, but most clones submitted to the OC collection probably have not been tested individually in an LR reaction. The LR reaction is usually quite efficient and robust. Possible causes of a very inefficient LR reaction (when the control reaction with the destination vector seems to work OK) include too little entry clone DNA in the LR reaction or an inhibitor in the reaction, such as from a comtaminant in the DNA miniprep. Mutations in the recombination junctions of the attL site also can lower efficiency, but mutations in the submitted clone should have been ruled out by the sequencing; and it would be rare (but not unheard of) for a mutation to arise during clone propagation that lowers reaction inefficiency. A large insert size (over 6-8 kb) can also have a modest effect on the reaction efficiency. Troubleshooting points would include: 1. Did the customer prepare his/her own DNA? 2. If so, how was the concentration verified? 3. If by spectrophotometer, did the 260/280 and 280/320 ratios look OK? 4. If the DNA is clean, mixing it with the control entry clone DNA in an LR reaction shouldn't significantly reduce the total colony count beyond that of the control entry clone colony count. 5. Picking another colony for DNA prep might get around a mutation arising during propagation, although this is a long shot. 6. Finally, even if the entry clone DNA is working at low efficiency, picking a few colonies should yield at least one good expression clone, assuming the background colonies are low, as they should be.

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