![]() ![]() Increase the number of amplification cycles in increments of 5, or, if possible, increase the amount of template.Ĭontaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Insufficient amplification can result if the initial amount of template is too low. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer. ![]() Use desalted primers or more highly purified primers. Try a control reaction in which you use a pure plasmid with the addition of the template to determine if any inhibitory effects exist.Ĭontaminants in primers may inhibit PCR. If inhibitors are suspected, dilute existing template otherwise, use fresh template and increase cycles. Template may be sheared or may contain PCR inhibitors. Template was damaged or degraded or contained inhibitors DMSO or another secondary structure destabilizer can be added (do not exceed 10%). For greater accuracy, optimize the annealing temperature by using a thermal gradient. To improve amplification, increase the annealing temperature. GC-rich PCR products are difficult to amplify. Each dNTP should be present at 200 μM in the final reaction.Įach dNTP should be present at 200 μM in the final reaction. If the dNTP concentration is too high, Mg 2+ depletion occurs. For the initial denaturation, use 3 min to activate the polymerase to denature the template during cycling, use 30 sec.īack to Top Causes Related to PCR Components If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will be low. For the initial denaturation, use 3 min at 95☌ for denaturation during cycling, use 30 sec at 95☌. If the denaturation time is too long, DNA might be degraded. If the denaturation temperature is too low, the DNA will not completely denature and amplification efficiency will be low. The annealing temperature should not exceed the extension temperature. If the primer T m minus 5☌ is close to the extension temperature (72☌), consider running a two-step PCR protocol. Use the lowest primer T m when calculating the annealing temperature. To calculate the primer T m, use the tool at with the default salt concentration and 0.2–1 µM primer (depending on your reaction conditions). The rule of thumb is to use an annealing temperature that is 5☌ lower than the T m of the primer. If the annealing temperature is too high, primers are unable to bind to the template. Use an annealing time of at least 30 sec. If the annealing time is too short, primers do not have enough time to bind to the template. Generally, use an extension time of 1 min/kb. If the extension time is too short, there will be insufficient time for complete replication of the target. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low. Using too few PCR cycles can lead to insufficient amplification. In conclusion, to determine the success of the PCR reaction, we need to identify the lanes in the gel image and analyze the bands in the gDNA extract, PCR amplicon lane.Causes Related to Cycling Times and Temperatures If there are multiple bands or no bands, then the PCR reaction may have been unsuccessful or nonspecific. If there is a clear band in Lane 3 that corresponds to the expected size of the PCR amplicon, then the PCR reaction was successful. Now, we can analyze the gel image to determine the success of the PCR reaction. Let's assume the lanes are labeled as follows: The gDNA extract, PCR amplicon lane should have one or more bands, depending on the success of the PCR reaction and the presence of any nonspecific amplification.īased on this information, we can identify the lanes in the gel image. The size ladder should have multiple bands with known sizes (1, 2, 3, 4, and 10 kb). The negative control should not have any bands, as it does not contain any DNA. Since we know that the lanes are (A) negative control, (B) size ladder (1, 2, 3, 4, and 10 kb), and (C) gDNA extract, PCR amplicon, we can look for patterns in the gel image to determine which lane is which. First, we need to identify the lanes in the gel image. ![]()
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