- v6 will include epitopes on the target protein (currently only Ty tags)
- v7 uses only the indicated protein tag.
- See the supplemental information in our Open Biology publication for the precise instructions on how to use these vectors.
- I have now switched to "Roditi" electroporation buffer and 3x 2000V in 4mm cuvettes - this has increased my transfection efficiency about 3 fold.
- Primers designed to tag a gene will work regardless of the drug resistance or the genetic tag.
- When preparing DNA containing drug ORF repeats, use a recombination deficient strain of E. coli such as sure2.
- Sequence files for older versions of pPOT can be found here. They have been replaced because they had lower transfection efficiency for C terminal tagging.
Notes on fluorescent proteins:
- mNeonGreen is a step change improvement from the other fluorescent proteins. It is better than eYFP or eGFP for genetic tagging in all respects (brighter, more photostable, increased proportion of 'good localisations').
- Superfolder GFP is optimised for tagging of membrane proteins. However, I have not personally tested that it works better than the other fluorescent proteins and I suspect that mNeonGreen still outperfoms it.
- tdTomato is very bright but frequently causes mis-localisation of the tagged protein. For this reason I would recommend tagRFPt as the current best red protein.
Dean, S., Sunter, J., Wheeler, R. J., Hodkinson, I., Gluenz, E. Gull, K. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol 5. 2015 (doi:10.1098/rsob.140197)Please contact me if you have any problems with the protocol or any of the files.