Plasmid Copy Number Estimator

This calculator converts between plasmid DNA mass (in nanograms) and copy number (number of molecules). Given the plasmid size in base pairs, it uses Avogadro's number (6.022 × 10²³ molecules/mol) and the average molecular weight of double-stranded DNA (660 g/mol per bp) to compute the conversion. It also provides molar equivalents in mol, fmol, and pmol. The calculations are: Molecular Weight = Size (bp) × 660 g/mol, Moles = Mass (g) / MW, Copies = Moles × Avogadro's Number. Understanding these calculations helps you convert between mass and copy number for ligation reactions, qPCR standards, and transfection optimization.

Double-stranded DNA consists of two complementary strands. Each nucleotide averages about 330 Da (Daltons), so a base pair (one nucleotide on each strand) totals approximately 660 Da. This is a well-accepted average for dsDNA calculations. The exact value varies slightly based on GC content (A, T, G, and C have slightly different molecular weights), but 660 g/mol per bp is accurate enough for most applications. Understanding this constant helps you see why molecular weight calculations are straightforward and why this value is used consistently in molecular biology.

The calculation is accurate for pure, intact plasmid DNA of known size. However, real-world accuracy depends on: (1) accurate concentration measurement (NanoDrop readings can vary with purity, Qubit is often more accurate for dsDNA), (2) correct plasmid size (verify from sequence or restriction map), and (3) DNA quality (degraded DNA or contaminants give less accurate results). The calculation assumes pure double-stranded DNA—contaminants, different plasmid forms (supercoiled vs. linear), or degraded DNA can affect accuracy. Understanding these factors helps you interpret calculated copy numbers correctly and know when to verify with additional methods.

Copy number (molecules) is useful for qPCR standard curves, viral particle calculations, and thinking about absolute quantities. Molar units (fmol, pmol) are more practical for enzymatic reactions like ligations and Gibson Assembly, where you mix fragments in specific molar ratios (e.g., 3:1 insert:vector). For example, in ligation reactions, you typically use 3-5× more insert than vector (molar), not mass. Understanding when to use each unit helps you work with the appropriate format for your application and achieve proper stoichiometry in enzymatic reactions.

First, calculate fmol or pmol for both your insert and vector using this tool. Then, to achieve a 3:1 molar ratio, use 3× as many pmol of insert as vector. For example, if you have 50 fmol of vector, use 150 fmol of insert. Convert back to ng if needed for pipetting. The key is using molar ratios (not mass ratios) because equal masses of different-sized fragments contain different numbers of molecules. Understanding this helps you achieve optimal ligation efficiency and avoid incorrect ratios that reduce cloning success.

The molecular weight calculation is the same regardless of DNA form (supercoiled, nicked, or linear all have the same molecular weight). However, supercoiled, nicked, and linear DNA can behave differently in applications like transfection or ligation. Supercoiled DNA often gives higher transfection efficiency, while linear DNA is preferred for in vitro transcription. For copy number calculations, form doesn't matter—the calculation assumes the same molecular weight. Understanding this helps you see that copy number calculations are independent of form, but form affects downstream applications.

Yes! The same formula applies to any double-stranded DNA. Just enter the fragment length in base pairs. For PCR products, use the expected amplicon size. The calculation assumes pure dsDNA. For example, a 1 kb PCR product uses the same calculation as a 1 kb plasmid fragment. Understanding this helps you use the calculator for any dsDNA fragment, not just plasmids, making it useful for cloning, qPCR, and other molecular biology applications.

This calculator is designed for double-stranded DNA (660 g/mol per bp). For single-stranded DNA, use ~330 g/mol per nucleotide. For RNA, use ~340 g/mol per nucleotide. These require a different calculator or manual adjustment. The key difference is that single-stranded molecules have half the molecular weight per base/nucleotide compared to double-stranded DNA. Understanding this helps you know when this calculator is appropriate and when you need different constants for single-stranded molecules.

NanoDrop (A260) measures all nucleic acids and can be affected by contaminants that absorb at 260 nm (RNA, proteins, salts). Qubit uses fluorescent dyes that bind specifically to dsDNA, giving more accurate measurements for pure dsDNA quantification. For plasmid calculations, Qubit values are often more reliable because they're specific to dsDNA and less affected by contaminants. Understanding this helps you choose the appropriate measurement method and interpret concentration values correctly for accurate copy number calculations.

qPCR standard curves often span 10¹ to 10⁸ copies per reaction. A common approach is to prepare a stock at 10¹⁰ copies/µL and make 10-fold serial dilutions. The specific range depends on your expected sample concentrations. For example, if your samples are expected to have 10⁵-10⁷ copies, prepare standards from 10¹ to 10⁸ copies to cover the range. Understanding typical copy number ranges helps you design qPCR standard curves that accurately quantify your samples.