Including ATG in cloning? - (Apr/11/2013 )
I'm planning to clone my gene into pCAG-EGFP so my gene would be fused with EGFP for visualization. The restriction sites are downstream of the start codon of EGFP. Should I include the start codon of my gene when designing primer? Or is it better to omit it? Or it doesn't matter? I have tried both in my previous cloning experiments and both methods seem to give expression, but I would like to know the proper method.
Any thoughts are much appreciated! Thanks!
People do either, it doesn't seem to make much difference. Technically you shouldn't need it and it could potentially cause some secondary structure changes when you have a tag on the N-terminus already, but in my experience it doesn't matter.
If you were using a shorter tag, such as FLAG or HA then there may be the problem of the ribosome skipping the ATG at the start of the tag and jumping straight to the ATG in your sequence, but I haven't seen this happen yet.