There are two steps involved in this calculation:
- The sample concentration is converted from ng/μl to nM.
- This concentration in nM is used as the starting concentration for a dilution step to obtain a solution at the target concentration
Assumptions: These calculations assume the sample is double stranded DNA and that the average mass of a pair of nucleotide bases is 660 Daltons (Da). This makes the formula weight* of the base pairs in the double stranded DNA 660 g. The inverse of the molecular weight is the number of moles of template present in one gram of material.
1. Convert ng/μl to nM:
The conversions factor and other inputs needed to perform these conversions are:
- Concentration in ng/μl (starting point)
- Strand length (in base pairs)
- 1 g = 1×109 ng
- 1 mole of nucleotide base pairs = 660 g
- 1 L = 1×109 μl
- 1 M = 1 mole/L
- 1 nM = 1×10-9 M
People with a solid understanding of the relationship between the units of ng/μl and nM can skip some steps, but the most comprehensive set of step between these two units is ng/μl > g/μl > moles/μl> moles/L == M > nM
The complete process looks like this:
2. Determine the dilution needed using C1V1 = C2V2
where:
- C1 = initial concentration in nM
- V1 = volume to add to dilution (unknown)
- C2 = desired concentration in nM
- V2 = desired volume in μl
solving for V
1 creates the following equation:
V1 will have the units of μl. It is the volume of the starting solution to add to the dilution. The amount of buffer equals:
μl of buffer to add = desired volume - V1
Example Solution: