We have currently trying to add a GFP tag to a gene buy using homologous recombination with a PCR product. The marker we are using is G418 resistance and the transformations are done by a normal lithium acetate protocol.
We are seeing several colonies appear on our selection plates, however, when we go to test our colonies by PCR (using primers outside of the gene that is being tagged with GFP), we are seeing a band appear the same size as WT instead of substantially larger as it is supposed to be if GFP has been attached.
Every colony we have tested has turned out this way in several rounds of transformations and several different genes that we have attempted to tag.
Any help that anyone has would be greatly appreciated!
Kaitlyn, G418 resistance does occur spontaneously in S. cerevisiae under selection pressure. How much G418 do you have in your plates, and do you repatch initial transformants on fresh G418-containing plates to make sure that they are truly G418-resistant?
Additionally, I am assuming you are using a kanMX cassette for this transformation. Does the strain you are transforming carry any other MX cassette for resistance to a different drug?
Hi Mark,
I think that must be what is happening. A rotation student in my lab is conducting this work and I'll have her re-streak the colonies on G418 to make sure they are truly resistant. We are using the kanMX cassette and the strains do not contain any other MX cassette. I'm thinking that for some reason the transformations just are not working and she is getting spontaneous resistance because she is not seeing very many colonies in the first place. Thanks for the help!
Try also to add the GFP tag in the other extreme (C-terminal or N-terminal). Some proteins are not functional with epitopes and can eventually release them. In this case, you would transform cells (you would be able to see colonies) but you would lose the tag
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