Hi,

I have been trying simultanious digestion of a modified pET24(+) plasmid with Spe1 and Nde1 in a single mixture. The buffers documentation states 100 % efficiency for Nde1 and 75 % efficiency for Spe1. An agarose gel test I did showed however that Nde1 is not cutting efficiently while Spe1 is cutting fine, and this prevents me from continuing with my biobrick assembly. The modification of the pET24(+) is that an IF1 gene with a Nde1 restriction site is incorporated.

I would now like to try to perform the digestion of the plasmid with the restriction enzymes separetly. I am not sure however on how to go about this;

Do I need to purify the DNA after the first cut to get rid of the first enzymes buffer before I move on with the next cut? Should I cut with Nde1 or Spe1 first? Any other general tips that you could give is more than welcome!

All the best!