The PCR cycle is composed of 3 major steps:
Denature (93-94°C). The PCR reaction requires a single-stranded template. The first step denatures, or melts, the double-stranded template DNA so that all the DNA is single-stranded.
Anneal (45-58°C). Once the template DNA has been denatured, the temperature is lowered to a level that allows the two oligonucleotide primers to anneal to the target segment of the template DNA.
Extension (72°C). The temperature is raised again to allow the Taq polymerase enzyme to add nucleotides to the 3'-end of the primer annealed to the template DNA.
1. As opposed to independently adding small volumes of reagents to each individual tube, we prepare a Master Mix solution in a 1.5 ml Eppi tube. This contains all reagents except the template DNA. Using the Master Mix will increase accuracy, reduce reagent loss on tips, and reduce tube-to-tube variability.
2. Below is a sample table for preparing Master Mix for 6, 50 ul reactions (including control) using 25ul of DNA template. The total volume was calculated using 7 tubes because there is usually slight loss of volume when the master mix is being aliquoted to individual PCR tubes.
There are endless variations regarding the reagent volumes, but the common reagent volume changed is the amount of sddH20. For example, if 1 ul of DNA template is used, then 32.75 ul of sdd H20 (as opposed to 8.8 ul) is used to still prepare a final 50 ul PCR reaction. In addition, other reagents (e.g. Mg, BSA, etc.) are sometimes added and once again the volume of sddH2O must be adjusted accordingly. A 25 ul total volume PCR reaction can also be prepared by cutting the volumes below by 1/2.
Master Mix (for 6, 50 ul PCR reaction):
|Volume (per tube)||Reagent||
|5.00 ul||10X Taq Buffer (+Mg)||
|1.00 ul||10X dNTP mix*||
|5.00 ul||Primer 1 (10uM)||
|5.00 ul||Primer 2 (10uM)||
|0.25 ul||Taq polymerase||
4. Add 25 ul of DNA template to each respective tube and mix gently with the pipet.
5. Cap the tubes and find an empty Thermal Cycler.
6. Talk to someone in the lab about how to
use the various Thermal Cyclers. Each one is slightly different,
but all have standard and modified PCR cycling programs already loaded.
Once you have determined the success of your amplification reaction, you can use the results to establish parameters which will optimize conditions for your primer-template combination. Optimization of the PCR protocol for each primer-template pair may be necessary and can be achieved by varying magnesium chloride concentration in the Taq PCR Buffer, primer concentration, dNTP concentration, and cycle conditions.
? The optimal magnesium chloride concentration can be tested empirically by titrating concentrations of MgCl2 for each primer set. The Taq PCR Buffer you used contains a MgCl2 concentration of 6.7 mM. This is much higher than most protocols recommend, therefore, you may want to reduce the MgCl2 concentration. Too little or too much MgCl2 could reduce amplification efficiency or result in non-specific products (mis-priming). If the samples contain EDTA or other chelators, raise the MgCl2 concentration proportionately.
? The optimal primer concentrations need to be determined empirically, by testing concentrations in the range of 0.1 to 1 mM. The primer concentration you used was 1 mM final concentration for the non-limited primer. Primer concentrations that are too low will result in little or no PCR product, while concentrations that are too high may result in amplification of non-target sequences.
? The concentration of dNTPs in the reaction mix should be well balanced. If the concentration of any one dNTP is significantly different from the rest, the polymerase enzyme will tend to misincorporate them, slow down, and terminate prematurely.
? The starting concentration of template DNA should be > 104 copies but less than 1 mg total DNA per 100 ml. Low concentrations of target DNA may require up to 40 cycles to produce sufficient product for analysis. The cycle parameters can be varied to optimize the conditions for denaturation, annealing, and extension. For problems with non-specific product, you may consider reducing the number of cycles to 25, however, you will also be reducing the quantity of your desired product. The following temperature cycles are variations you might consider based on your primer quality.
94-50-72 good or perfectly matched primers
94-48-68 poorly matched primers (>4 mismatches)
94-45-65 universal primer of questionable quality
Another cycling method which has solved problems with mis-priming, artifacts and Negative Control contamination, is the "Hot Start" procedure included in the Shields' Lab Manual. This method requires setting up the Master Mix with all components for PCR including template DNA, however, the buffered Taq polymerase is withheld until after one full heat denaturation run on the thermal cycler.
The effect of these variations can be monitored
by visualization of PCR product run on agarose gel and stained with ethidium
bromide. For further discussion, proceed to Section 4.