For anyone who has ever performed PCR, this problem is all too familiar. You’re expecting a successful reaction to occur within the first few cycles of PCR, and you see no evidence at all that anything’s happening. This can be incredibly frustrating! With so much time and effort invested in your project, you want to know that every step is being done correctly. If you don’t see an amplified product after multiple attempts with both high-quality template DNA and primers (as well as other factors), it can be tempting to give up on the idea altogether. But wait! That's not necessarily an option. Here are some tips for troubleshooting why your target doesn't appear in the first cycle:
You may have noticed that the target amplification product does not appear in the first cycle. Why is this? It's because there's no target sequence present in your template. A target sequence must be present for you to amplify it via PCR, as we discussed above. Because you're using a very small amount of template DNA (50ng), which will never result in more than one copy of your target sequence per reaction tube, you won't amplify any targets until after the first cycle has completed and DNA has been synthesized from your primer annealing sites.
The target PCR product first appears in the third cycle because of the presence of contaminating DNA and primer dimers. These contaminants are not amplified in the first cycle because they have a lower melting temperature than the target DNA.
Primers for PCR should be designed to bind to the target sequence. The binding site should be far enough from the 3' and 5' ends of the primer so that it does not restrict polymerase activity, but still close enough that it can catalyze extension of the DNA strand by one nucleotide.
The melting temperature (Tm) of a primer is also important in determining how well your PCR reaction will work. This is because once you have amplified your target sequence and are ready for denaturation and annealing, if your Tms do not match up correctly then there will be no product formed. To ensure this doesn't happen, make sure that both primers have around the same length, base composition and predicted melting temperatures so they can successfully bind to each other when mixed together during thermal cycling.
Another thing worth noting is that if you have chosen a pair of primers that are too long they may form intramolecular dimers which will inhibit amplification due to competition with other PCR products. Additionally, self-complementary sequences can cause problems during PCR if they're not sufficiently separated by bases on either side (usually at least 5bp).
To determine the optimal annealing temperature, you'll need to know the melting temperatures of both primers. The melting temperature is a measure of how stable a DNA polymerase will be when it binds to a specific sequence in your gene (or whatever gene you're amplifying). The higher this value, the more stable your PCR reaction will become. In order for your PCR reaction to work properly, it's important that all four separate strands bind together and form what we call “specifically bound complexes” with each other—these complexes are what make up our desired target product after three cycles of denaturation and annealing/extension.
Once these specifically bound complexes have formed between all four strands during each cycle of heat treatment (purification step), they can be denatured again by raising the temperature until one strand separates from another. This process allows room for another round of binding before starting over again through denaturing at 95°C and then lowering back down again so that another complex can form; this continues until all four strands have fully recombined into one very long molecule called double stranded DNA (dsDNA).
You will need to use a different concentration of primers depending on the size of your target sequence, and the length of your PCR product.
The optimal concentration is usually between 200-400 nM. This range allows for enough DNA template to be present in each reaction mixture, but not so much as to over-saturate with primer (which would result in lower yields). If you have a very short sequence that does not incorporate well into the final product, then you may need higher concentrations than this. However, if it does incorporate well into the final product then lower concentrations should be used so that you don’t run into issues where there are too many copies of one particular segment within your sample (this could lead to bias during sequencing).
Mg2+ is one of the most important ingredients in PCR. Too much Mg2+ can inhibit PCR, but too little Mg2+ can also inhibit PCR. The optimal concentration for successful PCR is between 1.5-4 mM.
BSA is a protein that, like the DNA template you're amplifying, has four nucleotides. In BSA's case, those nucleotides are adenine, cytosine, thymine, and guanine (A-C-T-G). Since these two molecules have similar properties to each other (they both have the same number of nucleotides), they will compete with each other for binding to the polymerase enzyme. The more BSA there is in your reaction mixture (at least in theory), the more likely it is that your polymerase will bind to it instead of your DNA template strand or primer strands.
This inhibition can be measured by how long it takes for amplification products to appear in a PCR run on agarose gel electrophoresis after adding BSA—or any other compound that competes with DNA binding sites—to your reaction mixture. In addition to having an effect on an assay’s time course results (i.e., how long it takes until you see amplification product), using too much non-specific competitor may also affect product size and yield as well as sensitivity because non-specific competitors can bind directly onto the surface of Taq ligase mix beads during reverse transcription which would prevent these beads from being washed away after reverse transcription finishes before PCR starts so fewer copies are made during PCR than otherwise would be expected based solely upon input serial dilutions; this phenomenon causes false negatives since their actual concentration could potentially exceed what we thought was going into our protocol but didn't actually get processed properly due some sort of error caused by improper reagent/solution handling during preparation steps such as extraction steps where #dna
To determine the optimal amount of template DNA for your PCR, you need to consider the following:
There are many reasons why we should use low amount of template DNA in PCRs. The first reason is that it reduces the risk of contamination. When using low amounts of template DNA, the chance that non-specific amplification will occur decreases. The second reason is that it increases specificity and sensitivity of PCR product. Using low amount of template DNA can increase both specificity and sensitivity because less template DNA means fewer copies being made after each cycle, which results in fewer products being present at any given time during the reaction; hence, lower background noise levels are achieved when amplifying small amounts of target molecules with high fidelity (low error rate).
In order to understand why you never see target amplification product in the first cycle, we need to look at what is happening on your template DNA during reverse transcription. Normally, as soon as a primer binds to a complementary sequence on your template strand, it will start copying its complement (i.e., synthesizing cDNA). As this happens, there is no longer any free template available for binding by another primer. The result is that no new primers will be able to bind while those already attached continue their work of copying their complements until they reach their terminus (end point) or are removed by heat denaturing of RNA-DNA hybrids that stick out from double stranded DNA when exposed to 95°C heat treatment after reverse transcription reactions (see Figure 1).
It is important to know the reasons why you may not see target amplification product in the first cycle. This will help you understand what might be wrong with your samples and how to fix it. If none of these tips helped solve your problem, we recommend contacting a professional who can assist with troubleshooting your PCR reaction