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Molecular Biology Lab Tools

14 free calculators for PCR, cloning, qPCR, restriction digests, ligations, sequence analysis, and more

Primer Tm Calculator

Calculate the melting temperature (Tm) of your PCR primers using multiple methods. Supports standard and nearest-neighbor calculations for accurate annealing temperature estimation.

Enter DNA bases only (A, T, C, G). Degenerate bases (R, Y, S, W, etc.) are accepted but will be estimated.

Results

PCR Master Mix Calculator

Calculate reagent volumes for your PCR master mix. Automatically adds 10% extra volume to account for pipetting losses. Supports standard PCR, qPCR, and RT-PCR setups.

Master Mix Recipe

DNA/RNA Copy Number Calculator

Convert between mass (ng) and copy number for DNA or RNA molecules. Essential for preparing standards for qPCR, digital PCR, and cloning experiments.

Enter mass to calculate copies, or enter copies to calculate mass

Result

Dilution & Reconstitution Calculator

Calculate dilutions using C1V1 = C2V2 formula. Perfect for preparing serial dilutions, working stocks, and qPCR standards.

Dilution Instructions

Serial Dilution Generator

Serial Dilution Series

PCR Troubleshooting Guide

Click on your PCR problem to see expert solutions with step-by-step recommendations.

No PCR Product (Blank Gel)

No visible band on gel after PCR amplification

Most common causes and fixes:

  • Check primer design: Verify primers match the template. Run BLAST to confirm specificity. Ensure Tm difference between forward and reverse is <5 C.
  • Optimize annealing temperature: Start 5 C below the lowest primer Tm. Run a gradient PCR (50-65 C) to find the sweet spot.
  • Template quality: Check DNA concentration with NanoDrop (A260/280 should be 1.7-2.0). Try increasing template amount (10-100 ng for genomic DNA).
  • MgCl2 concentration: Try increasing from 1.5 mM to 2.0-3.0 mM.
  • Increase cycle number: Try 35-40 cycles for low-abundance targets.
  • Check reagent expiry: Replace dNTPs and polymerase if older than 6 months after opening.
  • Positive control: Always run a known positive control to rule out reagent failure.

Multiple Non-Specific Bands

Extra unexpected bands alongside or instead of the target

How to improve specificity:

  • Increase annealing temperature: Raise by 2-5 C increments. Use gradient PCR to optimize.
  • Use hot-start polymerase: Prevents non-specific priming during setup at room temperature.
  • Reduce MgCl2: Lower from 2.5 to 1.5 mM to increase stringency.
  • Reduce primer concentration: Try 100-200 nM instead of 500 nM.
  • Touchdown PCR: Start annealing 10 C above Tm, decrease by 1 C per cycle for 10 cycles, then continue at optimal Tm.
  • Redesign primers: Use 18-25 bp primers, GC content 40-60%, avoid 3' complementarity, check for secondary structures.
  • Add DMSO or betaine: 2-5% DMSO helps with GC-rich templates.

Primer Dimers

Small bands (50-150 bp) appearing at the bottom of the gel

Eliminating primer dimers:

  • Redesign primers: Check 3' end complementarity between forward and reverse primers. Avoid runs of 3+ complementary bases at 3' ends.
  • Reduce primer concentration: Use 100-200 nM instead of 400-500 nM.
  • Increase annealing temperature: Raise by 2-3 C.
  • Hot-start polymerase: Essential for preventing room-temperature mispriming.
  • Decrease cycle number: Use only as many cycles as needed (25-30 for abundant targets).
  • Optimize template amount: More template = less primer dimer competition.

Smearing on Gel

Diffuse smear instead of a defined band

Common fixes for smearing:

  • Reduce extension time: Use 1 min/kb for Taq, 30 sec/kb for high-fidelity polymerases.
  • Lower template amount: Too much template causes non-specific amplification.
  • Reduce cycle number: Over-amplification degrades products.
  • Check DNA quality: Degraded template produces smears. Check on gel before PCR.
  • Optimize MgCl2: Excess Mg2+ reduces fidelity and specificity.
  • Fresh dNTPs: Degraded dNTPs cause incomplete extension.

Wrong Size Product

Band appears at an unexpected size on the gel

Diagnosing incorrect product size:

  • Verify primer binding sites: BLAST primers against the reference genome. Check for SNPs at primer binding sites.
  • Check for splice variants: If using cDNA, product may be smaller due to intron removal.
  • Genomic vs cDNA: Genomic DNA products include introns and will be larger than expected from mRNA sequence.
  • Pseudogenes: Primers may amplify a related pseudogene. Redesign with unique regions.
  • Verify ladder: Ensure molecular weight marker is correct and not degraded.
  • Sequence the product: Gel-purify and sequence to confirm identity.

Low Yield / Faint Bands

Target band is visible but very faint

Boosting PCR yield:

  • Increase template: Use 50-200 ng genomic DNA or 1-10 ng plasmid DNA per reaction.
  • Increase cycle number: Add 3-5 more cycles (up to 40 for rare targets).
  • Optimize annealing temperature: Lower by 2-3 C to improve primer binding efficiency.
  • Increase MgCl2: Try 2.0-3.0 mM for improved polymerase activity.
  • Extend extension time: Ensure sufficient time for complete synthesis, especially for long amplicons.
  • Use fresh reagents: Replace polymerase and dNTPs.
  • Add enhancers: BSA (0.1-0.8 µg/µL) or betaine (1-2 M) can improve difficult reactions.

Contamination (Negative Control Has Band)

Unexpected band in the no-template control (NTC)

Eliminating contamination:

  • Separate workspaces: Use dedicated areas for pre-PCR setup, DNA extraction, and post-PCR analysis.
  • Use filter tips: Always use aerosol-barrier pipette tips.
  • Fresh aliquots: Make single-use aliquots of reagents. Never return unused reagent to stock.
  • UV decontamination: UV-irradiate workbench, pipettes, and racks for 15-30 min.
  • UNG/dUTP system: Incorporate dUTP and treat with Uracil-N-Glycosylase before PCR to destroy carryover amplicons.
  • Change gloves frequently: Between handling template and setting up reactions.
  • New reagent batch: If persistent, make entirely fresh master mix from new stocks.

GC Content & Sequence Analyzer

Analyze any DNA or RNA sequence for GC content, base composition, molecular weight, extinction coefficient, and more. Supports FASTA format.

Accepts plain sequence or FASTA format. Non-ATCGU characters are automatically removed.

Sequence Analysis

Reverse Complement Generator

Generate the reverse, complement, or reverse complement of any DNA sequence. Essential for primer design, probe design, and sequence annotation.

Results

Molarity & Reconstitution Calculator

Convert between mass and molarity for oligonucleotides, proteins, and reagents. Calculate how to reconstitute lyophilized oligos to your desired concentration.

Oligo Reconstitution

Calculate the volume of water/TE to add to your lyophilized primer to get the desired stock concentration.

Found on the oligo spec sheet from your vendor (e.g., IDT, Sigma)

Reconstitution Instructions

Mass ↔ Molarity Converter

Convert between mass concentration (ng/µL, µg/mL) and molar concentration (nM, µM) for any molecule.

dsDNA: ~660 × bp | ssDNA: ~330 × nt | Protein: check datasheet

Conversion Result

Restriction Enzyme Digest Calculator

Find restriction enzyme recognition sites in your DNA sequence. Shows cut positions and expected fragment sizes for common enzymes used in cloning and analysis.

Select one or more enzymes. Common cloning enzymes are pre-loaded.

Digest Results

DNA/Protein Molecular Weight Calculator

Calculate the molecular weight of a DNA, RNA, or protein sequence. Provides detailed composition breakdown and extinction coefficient for proteins.

DNA/RNA: A, T/U, G, C only. Protein: standard single-letter amino acid codes.

Molecular Weight

qPCR Efficiency & Ct Analyzer

Calculate PCR amplification efficiency from standard curve slope, and perform relative quantification (ΔΔCt) for gene expression analysis.

Efficiency from Standard Curve

Enter the slope of your standard curve (log₁₀ dilution vs. Ct). Ideal slope is –3.32 (100% efficiency).

Typical range: –3.1 to –3.6

Efficiency Result

ΔΔCt Relative Quantification

Compare gene expression between treated and control samples, normalized to a reference gene.

Default 100% assumes perfect doubling. Use your calculated efficiency for accuracy.

ΔΔCt Result

Codon Table & DNA→Protein Translator

Translate a DNA coding sequence into its amino acid (protein) sequence. View codon-by-codon breakdown with the standard genetic code. Supports all three reading frames.

Enter the sense strand. Sequence should start with ATG for correct translation.

Translation Result

OD600 & Bacterial Growth Calculator

Convert OD600 readings to cell density, calculate doubling time from growth data, and determine dilutions needed for competent cell preparation or inoculation.

OD600 to Cell Count

E. coli default: 8×10⁸ cells/mL per OD600 unit. Adjust for your organism.

Cell Density

Doubling Time Calculator

Calculate generation/doubling time from two OD readings at different time points.

Growth Rate

Dilution to Target OD

Calculate volume to dilute a culture to a target OD600 (e.g., for induction at OD 0.4–0.6).

Dilution

Ligation Calculator

Calculate the optimal amount of insert DNA for ligation reactions based on your vector size, insert size, and desired molar ratio. Essential for molecular cloning.

3:1 is standard for sticky-end ligations. Use 5:1–10:1 for blunt-end.

Ligation Setup

Full Reaction Setup

Standard 20 µL T4 DNA Ligase reaction based on NEB protocol.

ComponentVolume
10× T4 DNA Ligase Buffer2 µL
Vector DNA— µL
Insert DNA— µL
T4 DNA Ligase (400 U/µL)1 µL
Nuclease-free H₂O— µL
Total20 µL
Incubate at 16 °C overnight (cohesive ends) or 25 °C for 10 min (quick ligation).