Hello Homa.

Oligomers or bigger nucleotides conjugated to or labelled with FITC or fluorophores by other means are easily excited and emit its fluorescence like very short burning fireworks under UV or laser activation. This is a contrast to that of antibody-FITC complex in which a comparatively huge protein with lots of tagging. The huge size seems to protect or be stable probably due to many FITC molecules bound, although even in this case prolonged activation by UV or laser abolish the fluorescence.

In particular, RNA molecules-FITC or other form are unstable and lost by a brief excitation in many reports, and many efforts are being made to overcome.

Regards.

Thanks a lot for all the answers.

Dear professor @Sang Ho Lee, You mean I should follow the light protection tips.

I think I have respected as as far as I could them like covering the tubes all the time and working in darkness.

Dear Dr. @ Frederick E Domann ,

Even if I have free FITC in solution, I should observe maximum absorbance of fluorescent molecule after PCR but I don't.

Thanks for the point

Dear Meral,

Actually I was talking about PCR product but I have checked them after exonuclease reaction too and no changing of fluorescent trait was observed.

Hello Homa.

When you describe 'final selected aptamers', what procedure is involved?

It would be inappropriate to compare the fluorescence directly between the FITC-primers solution (highly concentarted fluorescence) and amplified aptamers solution by PCR (much-diluted fluorescence), which you did not mention about length and molar ratio of FITC-ncleotide and PCRed fragment molecule.

Set the spectrofluorometer base line with non-fluorescent PCRed products (which you should do) and then measure the fluorescence of the fluorescent PCRed aptamers at the same concentration. Your concentrated FITC nucleotides and PCRed fluorescent aptamers cannot not be compared with their absorbance because one tube has FITC nucleotides, ant the other has totally different molecules that is amplified molecules, FITC-aptamers, before the fluorescence.

Could you just check the fluorescence of a 5ul dorp and a 5ul drop of PCR buffer alone on a glass slide under a fluorescent microscope (you need a quick view due to the decay upon the exposure)?

Best wishes.

Dear Prof. Lee,

I have compared fluorescence primer solution with PCRed fragments with a length of around 100 to 120 nucleotides after and before ethanol precipitation. But I have gained the same result for these concentrated and buffer free solutions. Anyhow it would be a good idea to change the blank solution and baseline correction.

According to your last suggestion, I will check fluorescence molecule emission in different buffers too.

Thanks for the great advice

Do you mean that the fluorescence can be detected, but only cannot be detected in flow? If so, is there any problem on the gating?

Dear Yubin ,

Actually it may happen because of inappropriate flowcytometer parameters setting but it's difficult to find a standard protocol for bacteria flowcytometry. That' s the problem . FYI we also observe variable auto-fluorescent emission area in each test that make us really confused.