Illumina Nm Calculator

nM Calculation Formula:

\[ nM = \frac{ng/μL \times 10^6}{660 \times length} \]

Concentration (nM):

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1. What is nM Calculation for Illumina Sequencing?

The nM calculation converts DNA concentration from ng/μL to nM (nanomolar) units, which is essential for Illumina sequencing library preparation. This conversion ensures accurate normalization of DNA samples for optimal sequencing results.

2. How Does the Calculator Work?

The calculator uses the formula:

\[ nM = \frac{ng/μL \times 10^6}{660 \times length} \]

Where:

\( ng/μL \) — DNA concentration in nanograms per microliter
\( 660 \) — Average molecular weight of a DNA base pair (in g/mol)
\( length \) — Length of DNA fragment in base pairs
\( 10^6 \) — Conversion factor (ng to g and μL to L)

Explanation: The formula accounts for the molecular weight of DNA and converts mass concentration to molar concentration, which is necessary for equimolar pooling of libraries.

3. Importance of nM Calculation

Details: Accurate nM calculation is crucial for Illumina sequencing as it ensures proper normalization of DNA libraries. This prevents over- or under-sequencing of samples and improves data quality and sequencing efficiency.

4. Using the Calculator

Tips: Enter DNA concentration in ng/μL and fragment length in base pairs. Both values must be positive numbers. The calculator will provide the concentration in nM units suitable for Illumina sequencing protocols.

5. Frequently Asked Questions (FAQ)

Q1: Why is 660 used in the formula?
A: 660 g/mol is the average molecular weight of a DNA base pair, which accounts for the different molecular weights of A-T and G-C base pairs.

Q2: What is the typical nM range for Illumina sequencing?
A: Most Illumina protocols recommend 2-10 nM for loading libraries, though optimal concentrations may vary by sequencer model and library type.

Q3: Does this calculation work for both dsDNA and ssDNA?
A: This formula is specifically for double-stranded DNA. For single-stranded DNA, use 330 g/mol as the average molecular weight per nucleotide.

Q4: Can I use this for RNA sequencing libraries?
A: For RNA, use 340 g/mol as the average molecular weight per nucleotide and adjust the formula accordingly.

Q5: How accurate is this calculation for mixed-base compositions?
A: The 660 g/mol value is an average that works well for most applications. For extreme GC-content samples, more precise calculations may be needed.