Copy number calculator for realtime PCR

This is version 2 of the calculator. The calculator available at the length tab is the same as the previous version. If you were using the previous version you won't see any difference in your results.

The Mass* tab allows users to enter an exact calculation for the mass of the sequence based on input from another calculator. If you have sequence data in fasta format, you can either enter "DNA* molecular weight*" into your favorite search engine or use the Polynucleotide Molecular Weight Calculator available here at SciencePrimer.com

If you have any questions you can reach out to me on twitter @scienceprimer or through the site's contact form.

Comments

31

Perfect, bookmarked! Could I have one for RNA as well?

The calculator now does ds and ssDNA* and ssRNA

Hi,

Thanks for writing this calculator!

I am trying to calculate copy number for an RNA virus.

However, should the mass* not be calculated using 340g/mole instead of 660g/mole since that is the average molecular weight* of a single RNA molecule? Unless one has to consider the dsDNA* that is formed during the PCR??

Any explanation would help clear my confusion... Thanks!

When ssRNA is checked, the calculator uses 349 g / mole in the calculation. 

When ssRNA is checked, should it be 340g/mole since the average MW for Ribonucleotide monophospates is actually 339.5?

I must have mistyped 9 instead of 0

Thank you ....this is so helpful.

 

I am glad you found it useful

Thanxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

For ssRNA, Does this formula consider 2.53x10E6 g/mol as the molecular size for genomic RNA?

This calculator converts from ng to number of copies based on template length. Is this what you are trying to do?

Hei,

thanks for calculator and explanation about calculation. 

I have a question. Above formula can be used for known template lenght. How can we calculate absolute A solution is formed when one substance is dissolved into another. In a solution, the solute is the material present in the lesser amount. In other words it is the minor component. Contrast with solvent: the material present in a greater amount.

There are many different media into which materials are be dissolved. Commonly water is the solvent and a solute is the material dissolved in the water.
"> A solution is formed when one substance is dissolved into another. In a solution, the solute is the material present in the lesser amount. In other words it is the minor component. Contrast with solvent: the material present in a greater amount.

There are many different media into which materials are be dissolved. Commonly water is the solvent and a solute is the material dissolved in the water.
" class="lexicon-term">* copy numbers from unknown sample. For example I have gene of intrest in plasmid of known length. I can get standard curve with dilutions and get slope and y-intercept etc. i am intrested in calculating number of copies of X gene in a sample of DNA* from mixture of bacterial colines from lets say food sample or water. I do not know it is genomic or plasmid DNA?

is there any formula to get copy number in such sample by comparing it to satandard of known template lenght.

regards

shaw

 

A DNA* sample of 100 molecular was amplified in a PCR instrument for 1 hour. Considering a cycle period* of 5 min in average, how many DNA molecular would you expect?
how to solve dis..>?

PCR is not a perfectly efficient process, so it is not possible to give an exact answer to this problem. This is why techniques such as real time PCR have been developed. 

If, however, we assume that the reaction is perfectly efficient and that the number of molecules doubles each cycle, then after 12 cycles (5 minutes per cycle for an hour) you'd have roughty (2^12) x 100 copies.  

This is so helpful, thanks a lot!

Good evening... I am attempting a PCR experiment where i am measuring cell free DNA*, my gene of interest is b-globin.... Several authors used a conversion factor of 6.6 to converst concentration* to copy number..... I am confused about 6.6, any idea what that is?

Thanks alot

Reham - The mass* of a DNA* nucleotide is about 330 Da (Daltons). This makes the mass of a base-pair 660 Da. The calculator above uses 660 in the process of converting ng and bp to copy number.

I am not sure why the authors you site use 6.6 instead of 660, but I assume a factor of 100 is accounted for somewhere else in their calculations.

Click on the 'About the calculation' above for more on how this calculator works

If I am using a qPCR and I get 1x106[] of DNA*, how can I calculate the genomic DNA in the sample if I don't know the amount of DNA nanograms. Is there a way to know that? The target is 200bp fragment to be amplified by qPCR while the whole genome of organism is 4419977bp. 

Is this copies/mL or copies/uL?

Hi Norman,  

This calculator takes a mass* of nucleotides* and strand length. It returns the total number of copies of the strands present in that amount of material. Concentration* depends on your sample. 

if you have DNA* at a concentration of 100 ng/ul. Then putting 100 in for mass along with the strand length will tell you the number of copies in 100 ng of material. Since the starting units were per ul, the final units will still be per ul (ie copies/ul).

In other words, concentration depends on thE sample.

Hope this helps. 

my gene of interest is 169bp in length. i have cloned it in to pgemt vector (3015bp). 

for an absloute quantification experiment in qPCR, i would like to use this plasmid as template.

so while calculating the copy number , so my doubt is  whether i should fill the column of 'length of DNA* strand' with  169 or 169+3015?

 

How about adding a feature to input (eg copy and paste) a specific sequence for the copy number calc ?

That will provide a more accurate MW for the calculation.

Hi Mark, 

That would be a nice addition to the current calculator. I'm curious, how much error do you think is introduced into your work by the use of a constant mw value across all 4 bases?

- ams

 

Chanced upon this website. So what is the meaning of a copy number calculator in layman terms? I am a student and got curious.

Hi,

an alternative to mark's suggestion would be a box for us to enter the mlecular weight* of the DNA* ourselves. We can obtain the data from cutting and pasting our sequences in other 3rd party programs which can more accurately calculate the mw. the difference can be up to 5-10% compared to the average mw using 650 or 660. 

 

Sue, I really like this suggestion. I will try to get this function added soon. 

 

** Update **

The site now has a tool for determining the molecular weight of a polynucleotide based on it sequence. Within the next few weeks I will update this calculator to accept these molecular weight values for calculating copy number. 

I have isolated the DNA* of my intersest .i dont know how to find the length of DNA. i am going to do PCR.

It is difficult to answer your quetion with the information you've provided. If you are doing a standard PCR off of genomic or plasmid DNA* you might not need to estimate the length at all. 

Hello,

I know the amount of total genomic DNA* my samples contained, but I want to figure out the copy number for a mitochondrial gene. Which genome length would I enter? The nuclear genome, mitochondrial, or a combination of both?

Thank you. 

MW of a DNA* bp should be 660 and not 330 as stated above.  (Or 650 if desired).  This is a typo above most likely.

The MW will be 660 (or 650) for double stranded DNA*. The calculator also allows you to calculate copy number for single stranded DNA in which case it uses 330.