PCR is a powerful technique for amplifying DNA, but it’s not always easy to optimize. In this post, I’ll discuss some ways to improve your PCR reaction by manipulating parameters like Mg2+ concentration and annealing temperature, as well as adding second pair of primers and increasing the number of cycles.
An important consideration is the initial denaturation temperature and time. This should be at least 95 C, but often it is 95 C for 30 seconds. It seems like this would be a good starting point, but there are some things you need to think about here. If your thermocycler doesn't have a separate heating block for the reaction chamber and there's only one way in which your DNA can get heated then it might not reach that temperature in 30 seconds.
Another issue is that if you're going to use primers with more than one nucleotide then they won't all melt at once so you'll probably want to consider giving them longer at higher temperatures (95 degrees celsius) before cooling them down again to carry on with PCR reactions!
If you suspect that the annealing of your primers may not be occurring at the expected temperature, or if there are mispriming sites on your template, increasing the annealing temperature will help. The optimal range is 60°C-65°C. If it is too low, you may not get any amplification; if it is too high, you may get incomplete amplification.
If you suspect that your primers may be working better at a lower temperature than the one indicated in your protocol, try lowering your annealing temperature by 2 to 5°C. This can help if you believe that the annealing of your primers may not be occurring at the expected temperature or if there are mispriming sites on your template.
A PCR has many parameters that can be adjusted to improve its yield and specificity. One way is to adjust annealing temperatures for each primer pair based on how many cycles have been run or on how well amplification looks after several cycles of PCR.
The Tm is the temperature at which a primer will anneal to its target DNA. For example, if you have two primers and one has a Tm of 50°C and the other has a Tm of 60°C, then your PCR won't work because the lower-temperature primer will anneal before the higher-temperature one does. This can create problems when trying to amplify your target sequence using those primers. To make sure both primers have similar Tms, add 1 μL of water to each tube (to make 100 uM) and heat with boiling water bath until dissolved completely. Then dilute each primer separately into 10 μL PCR mix (e.g., 25 mM MgCl 2 , 200 μM dNTPs; 0.2 mg/mL BSA).
After adding ~0.5–10 ng template DNA (depending on its size), incubate at 55–60°C for 15 min before adding 1 U Taq polymerase (or KOD Hot Start Polymerase) and go through steps normally used in PCR sequencing reactions
Another thing you need to consider is that sometimes a second pair of primers (internal primers) may be used at a later cycle number to accomplish full amplification of a large fragment, or to confirm its presence. You'll find this especially true if the DNA fragment size is too large for one pair of primers. The internal primer steps are often done at 25-30 cycles from the start point, but it can vary depending on what kind of reaction you're doing and what kind of temperature cycling protocol you're using.
In some cases, even after optimizing Mg2+ concentration and annealing temperature, there is still no band visible on a gel. In this case increasing the number of PCR cycles will amplify even faint bands on gels.
If you are having trouble amplifying your gene of interest with a particular primer pair try another one. You might want to include both forward and reverse primers in your experiment to see if they work better together or separately. You can also try primers designed by another manufacturer because some manufacturers are better than others at designing primers that work well in specific conditions (e.g., Promega).
There are many different types of kits that perform PCR reactions; each one has its own set of parameters and limitations so it is important to choose carefully which kit best suits your needs. It may be necessary to change kits if you find yours doesn't work well under certain circumstances such as low template amounts or high background noise levels due especially if these errors occur frequently during amplification reactions.
You can improve a PCR by manipulating the parameters of the reaction. These include:
I hope these tips help you improve your PCR.