Real time PCR

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Rio's picture
Real time PCR

I work on quantitative real time PCR.To get standard curve,I serially dilute my plasmid ranging from 109 to 10o dilution.But I don’t understand while finally assigning the copy no. after doin real time,what copy no. to enter  for each well.
My plasmid concentration is: 161.8ng/ul
So,the copy no.= 4.5 X 1010 Copies/ul
Now,I serially dilute it 10 fold to get
4.5x109, 4.5 x 108, 4.5 x107, 4.5x106….4.5 x 100 copies/ul
since I do assay for each in triplicate (10ul each well) I prepare 40ul reaction mix for each dilution.
SYBR green (2x)   20
primer  F                1
primer  R                1
Water                     16
                               38 + 2ul each dilution=40ul
Now what I don’t understand is what the copies in each well are.
Since my dilution was having 4.5 x 10 9 copies/ul
Now in a 40ul reaction mix it should be 4.5x109 x2 copies/ 40ul
                                                      (  x 2 as I take 2ul of plasmid in 40ul reaction)
so to get per ul,
                         4.5x109 x2 /40 copies/ul  
                       =.225 x 10 9 copies/ul
                       = 2.25 x 10 8 copies/ul
So,I believe my each well is having  2.25 x 10 8 copies/ul and I should assign the value 2.25E8.But is this right?. Please help me out as its urgent. My assays are working well but all my work gets struck at this point.
Thank you in advance.