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Standard curve using plasmid

Plasmid standard

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#1 thekid

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Posted 23 April 2012 - 07:38 AM

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

#2 detriar

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Posted 14 April 2013 - 05:10 AM

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 / [plasmid length in basepairs x 660]) 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

Edited by detriar, 14 April 2013 - 05:12 AM.





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