Standard curve

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emineanayol@yah...
emineanayol@yahoo.com's picture
Standard curve

Hi,
We bought Lihtcycler 480 our lab. I want to screen CaMV 35S promoter in RR soya. Forexample I have got %0.1 RR soya.My sample is 245,5 ng. If 1 ng soya DNA 598 kopy, My  calculate is 245x598= 146,809 , %0.1 x146,809=146 kopy. ?s ?t true? ?f is it true, How can I prepare standart curve. I want to prepare 100, 50,20,10,5 copy. How can I do.Would you like to respond me question to detail format.
 
Thank you.
Emine

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Emine,
When you say that your DNA sample is only 0.1% RR Soya DNA, do yo mean that out of a total of 245.5 ng only 0.1% of that DNA is RR soya? If that is the case, then your calculation sounds correct. 
Yet, in order to answer your question, we need more details. First you need to know the size of the RR Soya genome, and more importantly how many copies of the CaMV 35S promoter are present in one genome. Knowing the genome size and the number of copies of the amplicon you are detecting will enable you to correlate the amount of DNA to the copy number. A great resource for this kind of calculation can be found here.
 

emineanayol@yah...
emineanayol@yahoo.com's picture
Thanks your answer . But I

Thanks your answer . But I don't understand  that is.Forexample in my hand %0.1 RR soya . Soybean genome size 1.55x10^9 bp, genome copies in 1 ng soya DNA 598. I don't understand how many copies of the CaMV 35S promoter are present in one genome.%0.1 RR soya is my target. %0.1 RR soya is 245,5 ng. 245x598= 146,809 , %0.1 x146,809=146 kopy. ?s 146 copy CaMV?

Ivan Delgado
Ivan Delgado's picture
 

 
CaMV 35S is one of the most common promoters used in the transformation of plants. When this DNA is introduced into the plant, it typically integrates randomly through out the genome, so there could be many copies of it. The most common way of determining how many copies are present is to run a Southern blot. Once you know how many copies of CaMV 35S are present in one genome, you can prepare your standard curve. 
Alternatively, you can simply assume that one copy is equal to 1.55x10^9 bp of DNA (even if there are multiple CaMV 35S copies within one genome). The problem with that is that if you analyze another plant that has different numbers of CaMV 35S inserts, your quantification will be off. 
 

emineanayol@yah...
emineanayol@yahoo.com's picture
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Hi,Ivan.
 I prepared 100, 50,20,10,5, 2 copy of %0.1 RR soya. I use it for (CaMV) 35S and EPSP gene in soybean DNA. I used LC 480 but the show results only 100,50 and 10 copy. I want to smooth results. Can you help me?
 Thanks…
 Emine
 

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Emine,
What you got is a typical result: your assay can't detect less than 10 copies. Can you prepare DNA with a higher concentration? The ideal standard curve would be: 10, 100, 1,000, 10,000 and 100,000 copies. 
 

emineanayol@yah...
emineanayol@yahoo.com's picture
Hi, Ivan,

Hi, Ivan,

I have forgetten say that is, I prepared this concentration for LOD and LOQ. We have to see 5 kopy even one copy.I didin't prepare standart curve. What are you offer us?

Ivan Delgado
Ivan Delgado's picture
 

 
If your requirement is to detect down to 1 copy, then very likely you are going to have to re-design your assay. If an assay cannot detect below 10 copies, very likely you will not be able to optimize it to detect down to one copy. Most assays that are sensitive down to 1 copy are designed to detect repetitive sequences within the amplicon you are analyzing. For example the detection of E. coli DNA is done by detecting the 16S sequence, which is repeated a number of times in an E. coli genome.