Creating standard curve - (Jul/23/2008 )
I have lyophilised sequence with OD260=70.2nM. I do not know how to count the copies to creat standard curve. Please help me. This is the first time I use Realtime PCR.
to draw an accurate standard curve you can do as follows;
first you should convert the mass to molecules using the below formula (the amount of template should be expresseed in molecules):
Mass (grams) X avogadro's number
average molecular weight of a base X template length
eg. if 0.8 pg of a synthetic 75-mer single stranded oligonucleotide is used , we'll have:
0.8 X 10-12 X 6.023 X 1023
___________________________________ = 2 X 107 molecules (copies) of DNA
330 X 75
(for double stranded template you should use 660 instead of 330)
to generate a standard curve prepare seven 10-fold dilutios starting with 1X107 copies and ending with 10 copies. most instruments have softwares that draw standard curve but you can use excel too (drawing the log template as x value and Ct as the y value).
if you are using semiquantitative real-time you can easily prepare 4 dilutions of 100, 80, 40 and 20 ug/ml of template DNA and draw a standard curve.