Plan simple CRISPR reaction stoichiometry by calculating Cas nuclease and guide RNA stock volumes per reaction and for a master mix.
For Research Use Only. This planner calculates simple stoichiometry and does not optimize CRISPR conditions. Always follow manufacturer protocols and institutional guidelines.
Enter your reaction parameters and click Calculate to see results
Last Updated: November 20, 2025. This content is regularly reviewed to ensure accuracy and alignment with current CRISPR-Cas gene editing principles.
CRISPR-Cas systems use a Cas nuclease (like Cas9 or Cas12a) guided by a short RNA molecule (guide RNA or gRNA) to target specific DNA sequences for editing. CRISPR gRNA stoichiometry refers to the molar relationship between Cas protein and guide RNA in a reaction mixture. Proper stoichiometry ensures efficient complex formation between Cas protein and guide RNA, which is essential for successful gene editing. Understanding CRISPR stoichiometry is crucial for students studying molecular biology, biotechnology, gene therapy, and biochemistry, as it explains how to calculate reagent volumes, determine proper molar ratios, and prepare CRISPR reactions. Stoichiometry calculations appear in virtually every CRISPR protocol and are foundational to understanding gene editing experiments.
Molar ratios determine how many guide RNA molecules are present for each Cas protein molecule. Common gRNA:Cas ratios include 1:1 (equal molar amounts), 2:1 (excess gRNA to ensure complete Cas loading), and 3:1+ (higher ratios for specific conditions). The optimal ratio depends on experimental context, cell type, delivery method, and gRNA stability. Understanding molar ratios helps you see why stoichiometry matters—proper ratios ensure efficient RNP (ribonucleoprotein) complex formation and optimal editing efficiency.
C1V1 = C2V2 is the fundamental equation used to calculate stock volumes needed to achieve target final concentrations. This dilution equation relates initial concentration (C1) and volume (V1) to final concentration (C2) and volume (V2). For CRISPR reactions, you calculate Cas volume: V_Cas = (C_final × V_rxn) / C_stock, and gRNA volume: V_gRNA = (C_final × ratio × V_rxn) / C_stock. Understanding this equation helps you see how to convert between stock and final concentrations.
Master mix preparation saves time and reduces pipetting errors when preparing multiple reactions. A master mix is a pre-mixed solution containing Cas protein, guide RNA, and buffer that can be distributed across multiple reactions. Adding 10-15% overage accounts for dead volume in pipettes, tips, and tubes, ensuring you don't run short when aliquoting. Understanding master mix preparation helps you see why it's preferred for multi-reaction experiments and how it improves experimental reproducibility.
Practical considerations include RNP complex formation (pre-complexing Cas and gRNA before delivery), buffer composition (which depends on delivery method and cell type), stock concentration accuracy (measured by BCA/Bradford for protein, UV absorbance for RNA), and volume limits (ensuring calculated volumes don't exceed reaction volume). Understanding these considerations helps you design practical CRISPR protocols that are easy to execute accurately.
This calculator is designed for educational exploration and practice. It helps students master CRISPR stoichiometry calculations by determining Cas and gRNA volumes, calculating molar ratios, and preparing master mixes with overage. The tool provides step-by-step calculations showing how C1V1 = C2V2 is applied to CRISPR reactions. For students preparing for molecular biology exams, biotechnology courses, or biochemistry labs, mastering CRISPR stoichiometry is essential—these calculations appear in virtually every gene editing protocol and are fundamental to experimental success. The calculator supports multiple Cas types, helping students understand all aspects of CRISPR stoichiometry calculations.
Critical disclaimer: This calculator is for educational, homework, and conceptual learning purposes only. It helps you understand CRISPR stoichiometry theory, practice volume calculations, and explore molar ratios. It does NOT provide instructions for actual CRISPR procedures, which require proper training, sterile technique, safety protocols, and adherence to validated laboratory procedures. Never use this tool to determine actual CRISPR protocols, prepare CRISPR reactions for experiments, or make decisions about gene editing conditions without proper laboratory training and supervision. Real-world CRISPR involves considerations beyond this calculator's scope: gRNA design, off-target effects, cell type-specific requirements, delivery method optimization, and empirical verification. Use this tool to learn the theory—consult trained professionals and validated protocols for practical applications.
CRISPR RNP stoichiometry refers to the molar relationship between Cas nuclease and guide RNA in a reaction mixture. Proper stoichiometry ensures efficient complex formation between Cas protein and guide RNA, which is essential for successful gene editing. When Cas and gRNA are mixed at the correct ratio, they form a ribonucleoprotein (RNP) complex that can target and edit specific DNA sequences. Understanding stoichiometry helps you see why proper ratios are critical—too little gRNA leaves Cas unbound, while excess gRNA ensures complete loading and optimal editing efficiency.
Cas volume is calculated as: V_Cas = (C_final × V_rxn) / C_stock, where C_final is target Cas final concentration, V_rxn is reaction volume, and C_stock is Cas stock concentration. For example, for 1 µM Cas final in 25 µL reaction with 20 µM stock: V_Cas = (1 × 25) / 20 = 1.25 µL. Understanding this calculation helps you determine how much Cas stock to add to achieve the target concentration.
gRNA volume is calculated in two steps: (1) Determine final gRNA concentration: [gRNA]_final = ratio × [Cas]_final, (2) Calculate gRNA volume: V_gRNA = ([gRNA]_final × V_rxn) / C_stock. For example, for 2:1 gRNA:Cas ratio with 1 µM Cas final: [gRNA]_final = 2 × 1 = 2 µM. If gRNA stock is 100 µM and V_rxn is 25 µL: V_gRNA = (2 × 25) / 100 = 0.5 µL. Understanding this calculation helps you determine how much gRNA stock to add to achieve the desired molar ratio.
Molar ratios ensure proper stoichiometry in RNP complex formation. Common ratios include: 1:1 (equal molar amounts, may leave some Cas unbound), 2:1 (excess gRNA ensures complete Cas loading, common choice), 3:1+ (higher ratios for specific conditions or gRNA stability concerns). The optimal ratio depends on experimental context, cell type, delivery method, and gRNA stability. Understanding molar ratios helps you achieve optimal editing efficiency and avoid incomplete complex formation.
Buffer volume is calculated as: V_buffer = V_rxn - V_Cas - V_gRNA, where V_rxn is total reaction volume, V_Cas is Cas volume, and V_gRNA is gRNA volume. This gives the remaining volume needed to reach the target reaction volume. If buffer volume becomes negative, the chosen concentrations and ratio are incompatible with the total volume—you need more concentrated stocks, lower concentrations, or larger reaction volume. Understanding this calculation helps you verify that your stoichiometry is feasible.
Overage compensates for dead volume in pipettes, tips, and tubes. When distributing a master mix to multiple reactions, some volume is inevitably lost due to pipette tips, tube walls, and small pipetting variations. Adding 10-15% overage means preparing enough for 10-15% more reactions than you actually need, ensuring you don't run short when aliquoting the last reactions. Understanding overage helps you prepare sufficient master mix and avoid running out during distribution.
Typical concentrations vary by application: Cas stock (10-50 µM), gRNA stock (50-200 µM), Cas final (0.5-2 µM for most applications), gRNA:Cas ratio (1:1 to 3:1, commonly 2:1). These ranges depend on cell type, delivery method, and experimental goals. Understanding typical concentrations helps you choose appropriate values for your experiments.
This interactive tool helps you calculate Cas nuclease and guide RNA volumes for CRISPR reactions. Here's a comprehensive guide to using each feature:
Choose your Cas nuclease type:
Cas Type
Select: Cas9 (SpCas9, most common), Cas12 (Cas12a/Cpf1), or Other. This is for labeling purposes only—calculations are the same for all types. However, different Cas types have different gRNA structures, PAM requirements, and optimal conditions.
Enter how many reactions you plan to prepare:
Number of Reactions
Enter the number of reactions you plan to prepare. The calculator uses this to determine total volumes needed for master mix.
Enter the total volume per reaction:
Reaction Volume
Enter the total volume in µL per reaction. Typical ranges: 10-50 µL depending on application. This is the final volume after adding all components.
Enter your Cas and gRNA stock concentrations:
Cas Stock Concentration
Enter concentration in µM. This is typically measured by BCA or Bradford protein assay. Typical ranges: 10-50 µM.
gRNA Stock Concentration
Enter concentration in µM. This is typically measured by UV absorbance (A260). Typical ranges: 50-200 µM.
Enter your target Cas final concentration and gRNA:Cas molar ratio:
Target Cas Final Concentration
Enter the desired Cas final concentration in µM. Typical ranges: 0.5-2 µM for most applications. This depends on cell type, delivery method, and experimental goals.
gRNA:Cas Molar Ratio
Enter the desired molar ratio (e.g., 2 for 2:1 ratio). Common ratios: 1:1, 2:1, 3:1. The calculator calculates final gRNA concentration as ratio × Cas final concentration.
Enter overage percentage:
Overage
Enter overage percentage (default 10%). This accounts for dead volume in pipettes, tips, and tubes. Higher overage (15-20%) may be needed for multi-channel pipetting from reservoirs.
Click "Calculate" to get your results:
View Calculation Results
The calculator shows: (a) Effective reaction count (with overage), (b) Per-reaction volumes (Cas, gRNA, buffer), (c) Total volumes for master mix (Cas, gRNA, buffer), (d) Final gRNA concentration (calculated from ratio), (e) Notes and warnings.
Check Warnings
Review any warnings about negative buffer volume (incompatible concentrations/ratio) or other issues.
Example: Prepare 6 reactions at 1 µM Cas, 2:1 gRNA:Cas ratio
Input: 6 reactions, 25 µL/reaction, Cas stock 20 µM, gRNA stock 100 µM, Cas final 1 µM, ratio 2:1, 10% overage
Output: Per reaction: 1.25 µL Cas, 0.5 µL gRNA, 23.25 µL buffer. Total: 8.25 µL Cas, 3.3 µL gRNA, 153.45 µL buffer
Explanation: Calculator uses C1V1 = C2V2 to determine volumes, applies molar ratio to calculate gRNA concentration, accounts for overage in master mix.
Understanding the mathematics empowers you to calculate CRISPR volumes on exams, verify calculator results, and build intuition about RNP complex formation.
C₁V₁ = C₂V₂
Where:
C₁ = Stock concentration (µM)
V₁ = Stock volume needed (µL)
C₂ = Final concentration (µM)
V₂ = Final volume (reaction volume, µL)
Key insight: This equation relates initial and final concentrations and volumes. Solving for V₁ gives: V₁ = (C₂ × V₂) / C₁. Understanding this helps you see how to calculate stock volumes needed to achieve target final concentrations.
Determine how much Cas stock to add:
V_Cas = (C_Cas_final × V_rxn) / C_Cas_stock
This gives the volume of Cas stock needed to achieve the target final concentration.
Example: (1 µM × 25 µL) / 20 µM = 1.25 µL
Determine final gRNA concentration:
[gRNA]_final = ratio × [Cas]_final
This gives the final gRNA concentration based on the desired molar ratio.
Example: 2:1 ratio with 1 µM Cas → [gRNA]_final = 2 × 1 = 2 µM
Determine how much gRNA stock to add:
V_gRNA = ([gRNA]_final × V_rxn) / C_gRNA_stock
This gives the volume of gRNA stock needed to achieve the target final concentration.
Example: (2 µM × 25 µL) / 100 µM = 0.5 µL
Determine remaining volume for buffer:
V_buffer = V_rxn - V_Cas - V_gRNA
This gives the remaining volume needed to reach the target reaction volume.
Example: 25 µL - 1.25 µL - 0.5 µL = 23.25 µL
Given: 6 reactions, 25 µL/reaction, Cas stock 20 µM, gRNA stock 100 µM, Cas final 1 µM, ratio 2:1, 10% overage
Find: Per-reaction and total volumes
Step 1: Calculate effective reaction count
Effective Reactions = 6 × (1 + 10/100) = 6 × 1.10 = 6.6 reactions
Step 2: Calculate Cas volume per reaction
V_Cas = (1 µM × 25 µL) / 20 µM = 1.25 µL
Step 3: Calculate final gRNA concentration
[gRNA]_final = 2 × 1 µM = 2 µM
Step 4: Calculate gRNA volume per reaction
V_gRNA = (2 µM × 25 µL) / 100 µM = 0.5 µL
Step 5: Calculate buffer volume per reaction
V_buffer = 25 - 1.25 - 0.5 = 23.25 µL
Step 6: Calculate total volumes for master mix
Total Cas = 1.25 × 6.6 = 8.25 µL
Total gRNA = 0.5 × 6.6 = 3.3 µL
Total Buffer = 23.25 × 6.6 = 153.45 µL
Given: 10 µL reaction, Cas stock 5 µM, gRNA stock 10 µM, Cas final 2 µM, ratio 3:1
Find: Volumes and check feasibility
Step 1: Calculate Cas volume
V_Cas = (2 µM × 10 µL) / 5 µM = 4 µL
Step 2: Calculate gRNA volume
[gRNA]_final = 3 × 2 = 6 µM
V_gRNA = (6 µM × 10 µL) / 10 µM = 6 µL
Step 3: Calculate buffer volume
V_buffer = 10 - 4 - 6 = 0 µL (exactly at limit)
If V_buffer < 0, the stoichiometry is impossible—need more concentrated stocks, lower concentrations, or larger reaction volume.
Understanding CRISPR stoichiometry is essential for students across molecular biology and biotechnology coursework. Here are detailed student-focused scenarios (all conceptual, not actual CRISPR procedures):
Scenario: Your molecular biology homework asks: "How much Cas and gRNA stock do you need for 6 reactions at 1 µM Cas final, 2:1 gRNA:Cas ratio, with 25 µL/reaction, Cas stock 20 µM, gRNA stock 100 µM?" Use the calculator: enter the values. The calculator shows: Per reaction: 1.25 µL Cas, 0.5 µL gRNA, 23.25 µL buffer. You learn: how to use C1V1 = C2V2 to calculate volumes and apply molar ratios. The calculator helps you check your work and understand each step.
Scenario: Your gene editing lab report asks: "Explain why molar ratios (not mass ratios) are used in CRISPR RNP formation." Use the calculator: compare different ratios (1:1, 2:1, 3:1). Understanding this helps explain why proper stoichiometry ensures efficient complex formation, why excess gRNA improves Cas loading, and why optimal ratios depend on experimental context. The calculator helps you verify your understanding and see how ratio affects gRNA concentration.
Scenario: An exam asks: "You prepare a master mix for 6 reactions with 10% overage. What are the total volumes needed?" Use the calculator: enter 6 reactions, 10% overage. The calculator shows: Effective reactions = 6.6, Total Cas = 8.25 µL, Total gRNA = 3.3 µL, Total Buffer = 153.45 µL. This demonstrates how to account for overage in master mix preparation.
Scenario: Problem: "Compare gRNA volumes needed for 1:1, 2:1, and 3:1 gRNA:Cas ratios at 1 µM Cas final." Use the calculator: enter each ratio. The calculator shows: higher ratios require more gRNA volume. This demonstrates how molar ratio affects gRNA concentration and volume.
Scenario: Your biotechnology homework asks: "Why is proper stoichiometry important for CRISPR RNP complex formation?" Use the calculator: compare stoichiometry with different ratios. Understanding this helps explain how proper ratios ensure efficient complex formation, why excess gRNA improves Cas loading, and why stoichiometry affects editing efficiency. The calculator makes this relationship concrete—you see exactly how ratio affects gRNA concentration.
Scenario: Problem: "Your calculated buffer volume is negative. How do you fix this?" Use the calculator: try different concentrations or ratios. Understanding this helps explain why negative buffer volume indicates incompatible stoichiometry, and how to adjust concentrations, ratios, or reaction volume to make it feasible. This demonstrates how to troubleshoot stoichiometry problems.
Scenario: Your instructor recommends practicing different types of CRISPR stoichiometry problems. Use the calculator to work through: (1) Different Cas concentrations, (2) Different gRNA:Cas ratios, (3) Different reaction volumes, (4) Different stock concentrations, (5) Different overage percentages. The calculator helps you practice all problem types, identify common mistakes, and build confidence. Understanding how to solve different types of stoichiometry problems prepares you for exams where you might encounter various scenarios.
CRISPR stoichiometry problems involve dilution equations, molar ratios, and unit conversions that are error-prone. Here are the most frequent mistakes and how to avoid them:
Mistake: Using mass ratios instead of molar ratios for gRNA:Cas stoichiometry.
Why it's wrong: Molar ratios ensure proper stoichiometry in RNP complex formation. Mass ratios don't account for molecular weight differences between Cas protein and gRNA, leading to incorrect stoichiometry and poor complex formation. For example, equal masses of Cas and gRNA don't give equal molar amounts.
Solution: Always use molar ratios (gRNA:Cas) for stoichiometry. The calculator uses molar ratios—observe how it calculates final gRNA concentration from ratio × Cas final concentration.
Mistake: Using V = C × V instead of V = (C_final × V_rxn) / C_stock.
Why it's wrong: The correct formula from C1V1 = C2V2 is V1 = (C2 × V2) / C1. Using V = C × V gives wrong units and wrong volumes. For example, using 1 µM × 25 µL = 25 µL gives wrong volume (should be 1.25 µL for 20 µM stock).
Solution: Always remember: V_stock = (C_final × V_rxn) / C_stock. The calculator uses the correct formula—observe it to reinforce division by stock concentration.
Mistake: Using the same final concentration for gRNA as Cas, ignoring the molar ratio.
Why it's wrong: Molar ratio determines final gRNA concentration: [gRNA]_final = ratio × [Cas]_final. Using the same concentration for both gives wrong stoichiometry. For example, using 1 µM for both at 2:1 ratio should give 2 µM gRNA, not 1 µM.
Solution: Always calculate final gRNA concentration from ratio: [gRNA]_final = ratio × [Cas]_final. Then use this concentration to calculate gRNA volume. The calculator does this automatically—observe it to reinforce ratio application.
Mistake: Multiplying per-reaction volumes by actual reaction count instead of effective reaction count (with overage).
Why it's wrong: Master mix volumes should be calculated from effective reaction count (including overage), not actual reaction count. Using actual count gives insufficient master mix. For example, if you need 6 reactions but prepare for exactly 6, you may run out before finishing.
Solution: Always use effective reaction count for master mix: Effective = Reactions × (1 + Overage% / 100). Then multiply per-reaction volumes by effective count. The calculator does this automatically—observe it to reinforce overage calculation.
Mistake: Proceeding with calculations when buffer volume is negative, ignoring the warning.
Why it's wrong: Negative buffer volume means the combined volumes of Cas and gRNA exceed the total reaction volume. This is physically impossible—you can't have negative volume. The stoichiometry is incompatible with the chosen parameters.
Solution: Always check buffer volume. If negative, adjust: (1) Use more concentrated stocks, (2) Reduce target final concentrations, (3) Decrease gRNA:Cas ratio, (4) Increase reaction volume. The calculator warns about this—use it to reinforce feasibility checks.
Mistake: Mixing units (e.g., using mM when you need µM, or using µL when you need mL).
Why it's wrong: All concentrations must be in the same units (µM), and all volumes in the same units (µL). Mixing units gives wrong volumes. For example, using 20 mM instead of 20 µM gives 1000× too little volume.
Solution: Always check units: concentrations = µM, volumes = µL. Convert if needed: 1 mM = 1000 µM, 1 mL = 1000 µL. The calculator uses consistent units—observe them to reinforce unit consistency.
Mistake: Assuming the calculator recommends optimal Cas concentrations or gRNA:Cas ratios for your cell type or application.
Why it's wrong: This tool only calculates volumes based on your inputs. It doesn't optimize or recommend specific concentrations, ratios, or conditions for particular cell types, delivery methods, or applications. Optimal conditions vary widely and require empirical testing or manufacturer guidelines.
Solution: Always remember: this tool calculates volumes only. You must determine optimal concentrations and ratios separately (from literature, manufacturer instructions, or empirical testing). The calculator emphasizes this limitation—use it to reinforce that optimization is separate.
Once you've mastered basics, these advanced strategies deepen understanding and prepare you for complex CRISPR stoichiometry problems:
Conceptual insight: Molar ratios ensure proper stoichiometry in RNP complex formation. When Cas and gRNA are mixed at the correct ratio, they form efficient complexes that can target and edit DNA. Excess gRNA ensures complete Cas loading, improving editing efficiency. Understanding this provides deep insight beyond memorization: stoichiometry is fundamental to CRISPR function.
Quantitative insight: For a given Cas final concentration, higher gRNA:Cas ratios require higher gRNA final concentrations and more gRNA volume. This is because [gRNA]_final = ratio × [Cas]_final. Memorizing this pattern helps you quickly estimate gRNA volumes. Understanding this pattern provides quantitative insight into why ratio affects volumes.
Practical framework: Always follow this order: (1) Calculate final gRNA concentration (ratio × Cas final), (2) Calculate Cas volume (C1V1 = C2V2), (3) Calculate gRNA volume (C1V1 = C2V2), (4) Calculate buffer volume (V_rxn - V_Cas - V_gRNA), (5) Check feasibility (buffer ≥ 0). This systematic approach prevents mistakes and ensures you don't skip steps. Understanding this framework builds intuition about CRISPR stoichiometry.
Unifying concept: CRISPR is fundamental to gene therapy (correcting disease-causing mutations), biotechnology (creating cell lines, protein production), and research (functional genomics, disease modeling). Understanding CRISPR stoichiometry helps you see why accurate calculations are critical for experimental success, how proper ratios ensure efficient editing, and why optimization is essential. This connection provides context beyond calculations: CRISPR is essential for modern molecular biology.
Exam technique: For quick estimates: If Cas final = 1 µM and stock = 20 µM, Cas volume ≈ 5% of reaction volume. If ratio = 2:1, gRNA final = 2× Cas final. If gRNA stock = 100 µM, gRNA volume ≈ 2% of reaction volume for 2 µM final. These mental shortcuts help you quickly estimate on multiple-choice exams and check calculator results. Understanding approximate relationships builds intuition about CRISPR volumes.
Advanced consideration: This calculator provides volume calculations only. Real systems show: (a) Cell type affects optimal Cas concentrations, (b) Delivery method affects optimal ratios, (c) gRNA design affects editing efficiency, (d) Off-target effects depend on gRNA specificity, (e) Buffer composition affects RNP stability. Understanding these limitations shows why empirical optimization is often needed, and why advanced methods are required for accurate work in research, especially for novel cell types or experimental conditions.
Advanced consideration: Proper stoichiometry affects editing efficiency: (a) Too little gRNA leaves Cas unbound, reducing efficiency, (b) Excess gRNA ensures complete Cas loading, improving efficiency, (c) Optimal ratios depend on experimental context, (d) RNP complex stability affects delivery and editing. Understanding this helps you design experiments that use stoichiometry calculations effectively and achieve desired editing results.
• Volume Calculations Only: This planner calculates volumes based on the C₁V₁ = C₂V₂ relationship. It does not optimize or recommend specific concentrations, ratios, or conditions for particular cell types, delivery methods, or applications.
• Molar Ratio Interpretation: The tool uses molar ratios (gRNA:Cas) for stoichiometry calculations. Optimal ratios vary significantly depending on Cas variant, gRNA type, cell type, and experimental conditions—typically ranging from 1:1 to 5:1.
• Stock Concentration Accuracy: Calculations depend on accurate stock concentration values. Protein and RNA concentrations can degrade over time or vary between preparations, affecting actual stoichiometry.
• No RNP Complex Formation Verification: The calculator assumes that mixing at calculated ratios produces functional RNP complexes. Actual complex formation efficiency depends on incubation conditions, buffer composition, and component quality.
Important Note: This planner is strictly for educational and informational purposes only. It provides mathematical stoichiometry calculations for conceptual understanding, not actual CRISPR protocols. Experimental conditions, safety considerations, and institutional guidelines must be followed for any gene editing work.
The CRISPR-Cas gene editing principles and guide RNA stoichiometry concepts referenced in this content are based on authoritative molecular biology sources:
Optimal Cas:gRNA ratios (typically 1:2 to 1:5) are starting points. Empirical optimization is recommended for specific cell types and experimental conditions.
The gRNA:Cas molar ratio describes how many guide RNA molecules are present for each Cas nuclease molecule. A 2:1 ratio means there are 2 moles of gRNA for every 1 mole of Cas protein. Higher ratios ensure all Cas protein is loaded with gRNA, but the optimal ratio depends on your specific experimental conditions and should be determined from literature or manufacturer recommendations. The calculation is: [gRNA]_final = ratio × [Cas]_final. Understanding molar ratios helps you achieve proper stoichiometry in RNP complex formation and optimal editing efficiency.
No. This tool only calculates volumes based on the ratio you provide. It does not optimize or recommend specific ratios for your cell type, target gene, or delivery method. The optimal gRNA:Cas ratio varies by experimental context and should be determined through published protocols, manufacturer guidelines, or empirical testing in your system. Common ratios include 1:1 (equal molar), 2:1 (excess gRNA, common choice), and 3:1+ (higher ratios for specific conditions). Understanding this limitation helps you use the tool correctly and know when to seek additional optimization guidance.
When pipetting a master mix into multiple reactions, you inevitably lose some volume to pipette tips, tube walls, and minor pipetting variations. Dead volume is the liquid that remains in pipette tips, reservoirs, or tubes and cannot be dispensed. Adding 10-15% overage means preparing enough for 10-15% more reactions than you actually need, ensuring you don't run short when aliquoting the final reactions. For example, for 6 reactions with 10% overage, you prepare for 6.6 effective reactions. Understanding overage helps you prepare sufficient master mix and avoid running out during distribution.
No. This is a simple stoichiometry calculator that computes volumes based on concentrations and ratios. It does not account for cell type-specific requirements, delivery method (electroporation, lipofection, microinjection), transfection efficiency, or cellular uptake. Those factors affect protocol design but not the basic volume calculations. The calculator assumes simple C1V1 = C2V2 relationships and does not optimize conditions for specific applications. Understanding this distinction helps you see that volume planning and biological optimization are separate steps.
This indicates that the combined volumes of Cas and gRNA stocks exceed the total reaction volume at your chosen concentrations and ratio. You need to either: (1) use more concentrated stock solutions, (2) reduce the target final concentrations, (3) decrease the gRNA:Cas ratio, or (4) increase the total reaction volume. Negative buffer volume is physically impossible—the stoichiometry is incompatible with the chosen parameters. Understanding this helps you troubleshoot stoichiometry problems and adjust parameters to make the reaction feasible.
Cas9 (from S. pyogenes, SpCas9) recognizes NGG PAM sequences and creates blunt double-strand breaks. Cas12a (Cpf1) recognizes TTTN PAM sequences and creates staggered cuts. They have different gRNA structures and lengths. In this calculator, the Cas type selection is for labeling purposes only and does not affect the stoichiometry calculations. However, different Cas types may have different optimal concentrations and ratios in practice. Understanding this helps you know that calculations are the same, but optimal conditions may differ.
For Cas protein, use BCA or Bradford protein assays and verify activity. For guide RNA, measure concentration by UV absorbance (A260) using an appropriate extinction coefficient. Accurate stock concentrations are essential for reproducible experiments—inaccurate measurements will lead to incorrect molar ratios in your reactions. Always use freshly measured concentrations for accurate calculations. Understanding how to measure concentrations helps you ensure accurate stoichiometry calculations and successful CRISPR experiments.
Yes, the stoichiometry calculations apply to any reaction where you're combining Cas and gRNA at defined concentrations. However, in vitro cleavage assays may require additional components (target DNA, reaction buffer, Mg²⁺) that this calculator doesn't account for. The calculator only determines Cas and gRNA volumes—you must add other components separately. Always follow your specific assay protocol. Understanding this helps you use the tool for in vitro applications while recognizing its limitations.
All concentrations are in micromolar (µM) and volumes in microliters (µL). Make sure to convert your stock concentrations to µM before entering them. For reference: 1 µM = 1 µmol/L = 1 pmol/µL. Common conversions: 1 mM = 1000 µM, 1 nM = 0.001 µM. Understanding these units helps you enter values correctly and interpret results accurately. The calculator uses consistent units throughout—observe them to reinforce unit consistency.
No. This planner is strictly for research and educational use. Clinical applications of CRISPR require validated protocols, GMP-grade reagents, regulatory oversight, and extensive safety testing. This tool provides no clinical guidance and should not be used for any therapeutic purpose. Real-world clinical applications involve considerations beyond this calculator's scope: regulatory requirements, GMP compliance, safety testing, and validated procedures. Understanding this limitation helps you know when this tool is appropriate and when professional guidance is required.
Plan DNA/RNA transfection master mix volumes with overage calculations.
Convert plasmid DNA mass (ng) to copy number and molar equivalents.
Convert between mass, molarity, and concentration for DNA/RNA.
Calculate volumes and masses for buffer and reagent preparation.
Calculate Cas and gRNA volumes quickly for your gene editing experiments
Explore All Biology Calculators