Recombinant DNA

Question

Design a strategy for inserting your sequence of interest into the multiple cloning site so that the orientation of the insert is always the same

multiple cloning site includes SacI, smal, BamHI, EcoRI, HindIII

I don't understand how to do this.

Answer 1

Hi Vivien,

In this case you would pick any two of the restriction sites as there is only one blunt cutter in the mix (SmaI). As long as you have one site that creates an overlap when cleaved then the insert should be directional without issues. The other consideration here would be what temperature each enzyme cuts at, again here SmaI is not a good choice because it cuts at room temperature (25C). The last consideration is the compatibility of the buffers used for each enzyme, this will depend on the manufacturer. If you choose NEB enzymes for instance, then HindIII and BamHI can be double digested in buffer 3.1 or HindIII and SacI can be double digested in buffer 2.1. EcoRI is not a great choice because it requires a unique buffer and would require a sequential digest, and you can rule out SmaI due to the difference in temperature.

I think the best approach here would end up being a double digest using NEB enzymes HindIII and BamHI in NEB buffer 2.1 at 37 degrees C for one hour. Your insert would need to be flanked by the sites as follows: BamHI - insert sequence - HindIII (this is based on the assumption that the order is as listed above).

I hope this helps and it's not too late.