Having difficulties with your experiment?
We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios.
View the relevant questions below:
Here are possible causes and solutions:
- High background fluorescence due to riboflavin in the culture medium—replace medium with 1X PBS to eliminate background fluorescence.
- A filter set was used that did not allow excitation at the optimal wavelength or permit detection of the emitted fluorescence—use the XF76 filter from Omega Optical.
- Transfection efficiency is too low to allow detection of transfected cells—optimize your transfection conditions or try another method.
- Expression of Cycle 3 GFP may be low depending on the cell line used—in HEK-293 cells, maximum fluorescence was observed 72 hours post-transfection.
We would recommend checking to make sure that the gene of interest is cloned in-frame with the Platelet-derived Growth Factor Receptor (PDGFR) transmembrane domain.
If the gene of interest has a start codon in the context of a perfect Kozak sequence, it may be preferentially translated over the vector’s initiation codon, resulting in no leader sequence and no glycosylation, and hence no targeting to the endoplasmic reticulum and no secretion. This is rare, but it has been observed. If it is a problem, we recommend using PCR to delete the start codon.
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