Stay Tuned For Reagents, Protocols And Random Optical Components That We Construct In The Lab.
Resolving GFP from YFP (both optically and genetically)
A filter cube for imaging GFP without contamination from YFP: excitation: 452/25 dichroic: 484 LP emission: 494/20 All filters are available from Semrock for 25 mm cubes. This cube works well with various GFP strains in Cre/Ai32 (ChR2-eYFP) crosses for targeted patching of GFP cells and optogenetic stimulation of Cre-expressing axons (see Figure 7 in Banghart & Neufeld et al Neuron 2015). A decent camera may be required as the GFP signal is a bit dim relative to a standard eGFP cube. Although the excitation filter gets a bit of YFP, by omitting the peak of GFP's emission spectrum, the emission filter almost completely rejects any YFP signal.
On a related note, generic primers against GFP may give strong bands against YFP when the Ai32 transgene is present due to their high sequence similarity. We generated primers for GFP that do not recognize YFP: forward: CTG ACC TAC GGC GTG CAG TGC TT reverse: GCT CAG GGC GGA CTG GGT G product band ~ 500 bp Thermocycler protocol: Step Temp Time Note 1 94 3m 2 94 45s 3 66 45s 1.0*/cycle 4 72 45s 5 repeat steps 2-4 for 8 cycles 6 94 30s 7 58 30s 8 72 30s 9 repeat steps 6-8 for 19 cycles 10 72 10m 11 10 hold