Question in EGFP saw fluorescence but read zero in fluoremeter - (Aug/22/2008 )
Dear:
I expressed my EGFP under the normal lac promoter in E.coli DH5a induced by IPTG (1mM), under the fluorescence microscopy, I saw fluorescence. But when I took 200uL cell culture to 96 well plate without lysis, the reading in fuoremeter is zero. My explanation is that fuoremeter is less sensitive than microscopy, so fluoremeter can not detect the fluorescence.Contrary, I was told by my friend, whenever I can see in the microscopy, fuoremeter will give me reading because fluoremeter is much more sensitive than microscopy. Is that true? so, he said there is some mistakes during my process of my fluoremeter.If he is right, should I lysis the cell? And how much,by your experience, should I lysis the cell before fluoremeter?
Add more:
Could you please tell me what kinds of method to quantify the EGFP level expression. Which is the best in terms of sensitivity and linearity?
Thank you very much.
Hope to your help.
robbiegeng
I expressed my EGFP under the normal lac promoter in E.coli DH5a induced by IPTG (1mM), under the fluorescence microscopy, I saw fluorescence. But when I took 200uL cell culture to 96 well plate without lysis, the reading in fuoremeter is zero. My explanation is that fuoremeter is less sensitive than microscopy, so fluoremeter can not detect the fluorescence.Contrary, I was told by my friend, whenever I can see in the microscopy, fuoremeter will give me reading because fluoremeter is much more sensitive than microscopy. Is that true? so, he said there is some mistakes during my process of my fluoremeter.If he is right, should I lysis the cell? And how much,by your experience, should I lysis the cell before fluoremeter?
Add more:
Could you please tell me what kinds of method to quantify the EGFP level expression. Which is the best in terms of sensitivity and linearity?
Thank you very much.
Hope to your help.
robbiegeng
fluorimeter should be more sensitive than normal fluorescence microscope; may be you used the wrong plates which were not suitable for fluorescence measurement?