Real time_standard curve for plasmid dilution
No replies to this topic
Posted 27 December 2010 - 06:38 AM
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)
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 10e8,10e7,10e6Ö.10e0
According to formula c1v1=c2v2
=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 donít 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.