I am trying to clone/insert a second gene to the pRSFDuet1 vector but the second gene is not showing up in the gel after cloning. What precautions should be taken to successfully clone the second gene to the pRSFDuet1 vector?
Pierre Béguin
Provided that the first cloning went OK and that the restriction sites meant to receive the second insert are still preserved, no more precautions should be required than for the first cloning. There are a number of reasons why cloning can be unsucessful, but not more for duet cloning than for single cloning, except if the combination of the two genes turns out to be toxic. Should this be the case, a host affording stringent control of expression, like BL21 AI, could be helpful.
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