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PCR template concentration


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6 replies to this topic

#1 Moksha

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Posted 19 May 2010 - 11:40 AM

Hi there,

I am running a QPCR and with sybr green and I'm not sure how much template to use. I am going to titrate my template but am not sure what to use for my high-end titration point. My template stock concentration is 100um. Any advice?

Thanks!

#2 vladooo

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Posted 19 May 2010 - 12:27 PM

Use 10e8 or 10e7 copies as highest concentration. Then dilute in 10-fold steps.

#3 Moksha

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Posted 19 May 2010 - 01:08 PM

I'm sorry but I don't know what you mean....

#4 ProteinWork

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Posted 19 May 2010 - 02:54 PM

I'm sorry but I don't know what you mean....

10e8=100,000,000
10e7=10,000,000

#5 Moksha

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Posted 19 May 2010 - 03:33 PM

I understood the exponential terms, what I don't understand is what is meant by "copies".

:wacko:

#6 vladooo

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Posted 20 May 2010 - 12:08 AM

One copy is one molecule of template DNA.
What kind of template you have? Plasmid? cDNA? PCR product?
What is your template concentration? I don't think it is 100 uM. You were talking about primers probably.

#7 pop09

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Posted 20 May 2010 - 03:10 AM

Hi, if you are not sure how much of the template youwant to use, why not perform a 10-fold serial dilution of your template to check not only the efficiency of your run but also the good concentrations you can work with in succeeding runs. By the way, what knd of template are you working with? same questions as the one posted by vladoo.




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