Hello friends.
I am optimizing the method for estimating gene copy index by using Beta actin as house keeping gene. In each trail using same genomic DNA, I am getting different CT value. Can any one help me please how can I troubleshoot the problem.
And one more thing is, every day i used to check DNA concentration, i can see fluctuation in DNA concentration too. How can I prepare stable DNA?
How much fluctuation? How much CT value variation?
A simple test would be to use SEVERAL genomic amplicons: if these vary, but all vary to exactly the same extent, then your methodology remains essentially solid.
Maybe you can use plasmidic DNA as external standard to increase the stability
Dear John
For the same genomic DNA only am getting variation when I did multiple trails.
1. Do the standard curve assay by using the same DNA sample with serials dilution and then you will get a reasonable linear range.
2. Make sure the concentration of your sample at the linear range.
3. Others, primers with good amplification efficiency; a reasonable Ct value (early 20s) ect.
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