question on gateway - (Feb/01/2014 )
Does anyone here work with gateway technology?
If so: would it be best to introduce the gatewaysequences after creating the ideal vector or the other way around and make it gatewaycompatible first and then introduce the C and or N tags?
The latter seems harder because I have a gateway vector however, I dont have restriction sites to cut the vector anymore after the gateway sequences. Or is there another way to cut the vector?
Do what is simplest.
You can use PCR to amplify your desired DNA sequence with primers that contain the restriction site that you would want to use. You can do this with the entire vector too. Most high fidelity proof reading DNA polymerase can amplify 5kb without optimization nowadays.
You can add the att sites via PCR with long primers (~200bp).
Given that gateway vectors then to be used with recombinase in mind, there aren't that many useful sites in these vectors. So it would tend to be easier to do all your building first in some plasmid then transfer your completed structure to the gateway clone.