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[renz]

Hi all,
I am new to real time PCR and wud really appreciate if someone helps me thru this.
size of insert :309bp
Plasmid:2974 hence, total size:3283
mass of plasmid= (3283)(1.096*10e-21)

                       3.5*10e-18g


I used this to calculate 10e9 to 10e0 copies.

for 10e9 =3.5*10e-18*10e9=3.5*10e-9

similarly, I did calculation for 10e8,10e7,10e6….10e0


Conc:111.9ng/ul
      ie 1.1*10e-7g/ul
    
According to formula c1v1=c2v2
                    =3.5*10e-9*100/1.1*10e-7
                    =3.2ul plasmid + 96.8 ul nuclease free water

this is the calculation for 10e9 diln,similarly other dilutions were made with 2ul of plasmid and 18ul d/w


Then I used 2ul of each dilution in 20ul pcr mastermix(SYBRGREE),and added sample 10ul in duplicate.
I usually don’t get standard curve after 10e6 dilution.Am I doin any thng wrong in my calculation?What is the use of copy no. in this?I got 3.13*10e10.