Standard curve using plasmid - (Apr/23/2012 )

Hello.

I am doing bacterial copy quantification using a plasmid as my standard. The plasmid has a 1356bp fragment that is cloned into a topo vector of 3796bp to give a final plasmid size of 5152bp.

This plasmid is used to quantitative real time pcr to determine the copy number of bacteria present using TaqMan chemistry on a ABI 7500 machine.


I would like to know how to determine the copy number of plasmid present at the end of the plasmid miniprep and further dilutions to get a standard curve from 10⁸,10⁷,10⁶,10⁵,10⁴,10³,10²,10¹ copies of the plasmid.

regards,

thekid

Hi thekid, I'll try to answer ur question but since I'm also a newbie in this field, you have to see for answers from others.

Here's a way to determine your copy number of plasmid, I quoted it from Qiagen protocol:

After measurement of plasmid DNA concentration (using the spectrophotometry), the copy number of standard
DNA molecules can be calculated using thisformula:

(X g/μl DNA / ) x 6.022 x 10^23 = Y molecules/μl

for the serial dilution, after you determine the plasmid copy number (let's say the 10^8 is the undiluted sample), then you can simply following this step:
-Pipette 18 μl of Nuclease-free water into 7 microfuge tubes
-thaw your sample (tube 1), mix well,pipette 2 μl into tube 2. Pipette up and down to mix.--> this is 1:10
-using a new tip, pipette 2 μl from tube 2 to tube 3. Pipette up and down to mix.-->this is 1:100
-repeat the same process for tubes 4--7.
and that is, you make your serial dilution.

but, as I told you above, I'm new in qPCR so you have to see others' opinions.

Cheers,
Dee