madelingirly

Dear Guys,
Does any one have some experience in the use of softwares for quantitative comparison of images taken using fluorescence microscope.
I have no experience using these software ever.
what is the best and easiest software?
I wonder if u could share ur experience with me, or share any books or papers for beginners like me?
Thanks in advance guys
Madelin

Dear Guys,
Sorry for this silly question, but I need ur help
I know what is the meaning of Standard error and standard deviation.
and how to calculate them.
My question is, if my results would be expressed as % for example from some kind of equation.
How I can calculate SD or SE.
lets say this, the number obtained from my individual samples would be 5, 6, 5.5, 7.
and I compare it with control gives number of 10.
so if my equation is mean/control X 100
when I put it in my equation, the mean results would be expressed as % so results would be 58%

However, if I calculate SD or SE, from these numbers 5, 6, 5.5, 7. it will be so small (0.85) compared to result and incomprehensible?
How to calculate SD, or SE for results expressed from equations
Madelin

Dear Guys,
I all new in reverse transcription-PCR, I need ur advice.
I read that in reverse transcription-PCR results, the test with low Ct values this means the presence of starting material then, it is more positive.
but when I saw the papers, I can see that they express their results related to standard gene like GADH.
what was in my mind:
I will divide my Ct value of test to Ct value of STD gene,
the I get a number, I will use this number and put it in my graph.
My first question, Does this is ok??
but when I did this, I got one test has value of 2
and other test has value of 1.3
So I guess this means that first one is expressing my gene more that the second one.
but when I think that higher values means less expression as I am using Ct value?? I get confused.
So please help
Second issue.
how I can get Standard deviation  (STD) or Standard error (STE) for this results?
Divide STD from tested gene samples to STD of standard gene??
or what I can do
Thanks Guys for ur help

Dear Guys,

I am new in PCR, I am using Bioapplied prism 7000

Today was my third repetition for the same experiment.
My first time, I was in a hurry, so closed the machine and program after finish without looking to the results. Next day, I did not find any good amplification plots, small irregular curves.
Ok I said it is my first time, lets repeat it again.
So I repeated it, again, the same experiment, after finish, I looked at the amplification plots and it was there, regarding melting curve it was fine not perfect but ok.
then I closed PC, and machine.
However, next day I did not find what I saw, no amplification plots at all, small irregular curves..
I asked my lab-mates if they faced a similar problem with that machine.
they said no.
SO I said ok, perhaps I am a little bit crazy,, but not to the delusions level, any way I would repeat it again.
Then I repeated the experiment today again.
I looked at the amplification plots and it were there for the tests.
Then close the program, ok, and reopen it, they are gone!!!!
small irregular curves are there only for tests.
Please I am gonna be mad,,, where are my curves??
does any one have a similar experience.

Did I miss something during machine preparation(I made my lab-mate track all my steps to set machine after second experiment)?? If I did not save my experiment, the machine will not start measuring.

what I do for analysis, I just select my wells in plate then click on results, and click on amplification plots and it is there??

Another thing, when I tried to see Standard curve I could not??? during my first observation, so there were good amplification plots but no standard curve at all.
after close and open, I use only 6 STD, how I can see 8 red points in standard curve??
please help me as I will be crazy????
when I close the exp, I said dont save, so it should be the original one??
is it possible that machine show me false results after the experiment?? but when I close program reopen, the result is true??
Please I need some opinion of this machine user
Please help or I will be mad, and I am already half mad today.
Madelin

Dear Guys,
I tried to read more to understand Rt-PCR, to perform a good reliable experiment.
I read about melt curve and it helps u to identify unspecific primer-dimer.
and I saw an example.
but let me say my question according to the example,
Now I generated my melt curve, it will show decrease in fluorescence as function of temp.
so if I have 2 peaks??? according to definition of Tm, it is temp which 50% of dsDNA bases have been denatured??
so how I can know which one is my true Tm.
or just finding 2 peaks is enough to say, I have unspecific product, lets repeat the experiment in another way???

I am very grateful for those who is welling to help me with their experience
Best Regards
Madeline