What concentration of primer should I use for PCR?
Table of Contents
What concentration of primer should I use for PCR?
between 0.1 and 0.5 µM
The concentration of each primer should be between 0.1 and 0.5 µM. For most applications 0.2 µM produces satisfactory results. Too high primer concentrations increase the chance of mispriming, which results in nonspecific PCR products. Limiting primer concentrations result in extremely inefficient PCR reactions.
What is the final concentration of the dNTPs in a 25 μl PCR reaction as shown in the table?
Protocol
| Component | 25 μl reaction | Final Concentration |
|---|---|---|
| 10 mM dNTPs | 0.5 µl | 200 µM |
| 10 µM Forward Primer | 0.5 µl | 0.2 µM (0.05–1 µM) |
| 10 µM Reverse Primer | 0.5 µl | 0.2 µM (0.05–1 µM) |
| Template DNA | variable | <1,000 ng |
How do you optimize PCR primers?
Design both primers to have melting temperatures within 3°C of each other to simplify your PCR optimization. End with a G or C. Capping the 3′ end of your primer sequence with a G or C will strengthen primer annealing at the site of extension. Remember to add spacers for restriction enzyme cloning/isothermal assembly.
How much template should I add to PCR?
Even though in theory, one molecule of the template would be sufficient, considerably larger amounts of DNA are typically used for a classic PCR, for example, up to 1 µg of genomic mammalian DNA and as little as 1 pg of plasmid DNA (1).
How do you calculate PCR mix?
Add equal volume (eg. 10 ul) of of 1 mM DIG-dUTP. THEN add water to 10 vol (=100 ul; add 51 ul): final concentration each dNTP = 1 mM; final concn DIG-dUTP = 0.1 mM, and of dTTP = 0.9 mM.
How do you set a PCR cycle?
After the initial denaturation step, subsequent PCR cycles begin with a separate denaturation step that lasts 0.5–2 minutes at 94–98°C. As with the initial template DNA denaturation step, the time and temperature should be optimized according to the nature of the template DNA, DNA polymerase, and buffer components.
How do you calculate DNA volume in PCR?
Use the C1V1 = C2V2 volume formula, (C = concentration, V = volume) but you must use the same units for each. You’ll have to dilute a lot your DNA so I recommend you make an aliquote of a lower concentration (Ex.
How do you create a PCR program?
When designing primers for a PCR assay, follow these steps:
- Check the literature and databases for existing primers.
- Choose a target sequence.
- Design primers.
- Check primer specificity.
- Assess primer properties (melting temperature [Tm], secondary structure, complementarity).
- Determine PCR product properties.