How to calculate moles of DNA from base pairs.?.explain the concept.
Answers
A few key points to remember :
● The average weight of a single DNA base pair (bp) is 650 daltons. This can also be written as 650 g/mol (= molar mass)
● This is the same as saying that one mole of a bp weighs 650 g
● The molecular weight or molar mass of any double stranded DNA fragment can therefore be calculated by multiplying its length (in bp) by 650 and the answer will be expressed as daltons or g/mol.
Let’s look at an example:
Question: What is the molecular weight of a 5.2kb plasmid? Remember 5.2 kb = 5,200 bp
Calculation: 5,200 bp x 650 daltons = 3,380,000 daltons or g/mol
Now we know how to calculate the molecular weight of a DNA template. Using Avogadro’s number, which is 6.022×1023 molecules/mole, the number of molecules of the template per gram can be calculated using the following formula:
ng is the amount of DNA (plasmid, primer etc.) you have in nanograms
6.022×1023 = Avogadro’s number
length is the length of your DNA fragment in base pairs. Just multiply by 1000 if you are working in kb.
We multiply by 1×109 to convert our answer to nanograms
Note: the Avogadro’s number or constant (denoted as L, NA) is defined as the number of constituent particles (usually atoms or molecules) per mole of a given substance
Now let’s try out the formula using a few examples
Example 1: Plasmid
Question: You have 300 ng of a 5.2 kb plasmid.
How many copies of your plasmid do you have?
Calculation: Number of copies = (300*6.022×1023)/(5,200*1×109*650)
Example 2: Primer
Question: You have 50 ng of a 20 bp primer.
How many copies of your primer do you have?
Calculation: Number of copies = (50*6.022×1023)/(20*1×109*650)
Example 3: PCR product
Question: You have 150 ng of a 670 bp purified PCR product.
How many copies of your PCR product do you have?
Calculation: Number of copies = (150*6.022×1023)/(670*1×109*650)
Note: It’s important to remember that this formula is based on the assumption that you are working with a single DNA species, so when applying to PCR products or plasmids, make sure you only have 1 band or that you have a clean plasmid miniprep.