5 µl of this solution can be used as PCR template for re-amplification.) (A small plug of the gel can be removed with a micropipette tip, and the DNA can be recovered by adding the plug to 200 µl of water and then incubating at 37☌. Instead, the PCR conditions will need to be optimized consider the following when adjusting the PCR conditions: If the negative control is blank, there is no contamination. This can determine if the cause of the smear is contamination or overcycling, or if the smear results from poorly designed primers or suboptimal PCR conditions. If PCR generates a smear after running the products on a gel, what can be done to improve the results?įirst, determine the source of the smear using positive and negative (no template) controls. If PCR yield is low, try increasing the number of cycles by 5. The general recommendation for extension time for this enzyme is 10–20 sec/kb. Solution:Įxcessively long extension times may result in smearing. Smearing of the PCR product bands on a gel. The hot-start versions of these enzymes may improve results for some primers. Nonspecific primer annealing at low temperatures. Takara Ex Taq and Takara LA Taq DNA polymerases Issue: If the primer T m values are <55☌, try a shorter extension time between 5 and 10 sec/kb. To amplify targets 55☌, and use an annealing temperature of 60☌. To achieve specific amplification, it is essential to use a short annealing time (5–15 sec) when performing three-step PCR. PrimeSTAR HS and PrimeSTAR Max DNA polymerases Issue: Increase the annealing temperature in increments of 2 degrees.PCR conditions are not sufficiently stringent. Redesign primers if necessary or modify PCR conditions. Use BLAST alignment to determine if the 3' ends of the primers are complementary to sites other than the target site(s). If there are nonspecific amplification bands, what can be done to improve specificity? Although the standard extension time is 10 sec/kb, the extension time can be increased to ~0.5 min/kb for complex templates such as human genomic DNA. When using SpeedSTAR HS DNA Polymerase, consider: If the amount of template exceeds 200 ng in a 50-µl reaction mixture, set the extension time between 30 sec/kb and 1 min/kb. Adjusting the extension time if the reaction mixture contains excess template.When using PrimeSTAR Max DNA Polymerase, consider: Increasing the concentration of the primers.Using an extension time of at least 1 min/kb.If the template is human genomic DNA or a cDNA library, use no more than ~100 ng of the template in a 50-µl reaction mixture. Using an appropriate amount of template.When using PrimeSTAR HS DNA Polymerase, consider: Also, consider re-amplifying the primary PCR product using 10-fold dilutions (1:100 to 1:10,000) using nested primers. Visit our PCR selection guide to find an appropriate enzyme.Ĭheck your primers carefully redesign if necessary. When amplifying from templates with high GC content, use an enzyme formulated for this condition. If purifying the template is not a possibility, an enzyme that has a higher tolerance to impurities, such as Terra PCR Direct polymerase, may improve results. Alternatively, the template may need to be purified using a kit such as the NucleoSpin Gel and PCR Clean-up kit. If PCR inhibitors are present, using diluted template may increase PCR efficiency.
Refer to the guidelines provided with the enzyme to determine the optimal amount of template.Ĭonsider these additional possible reasons for PCR failure: Lower the annealing temperature in increments of 2 degrees.Consider modifying the PCR conditions as follows: If increasing the cycle number does not improve results, the PCR conditions might be too stringent for the particular primer set or template.Increasing the cycle number can overcome issues with a low-abundance template or template inaccessibility due to impurities in or poor priming efficiency of the primers. If there were no problems with the experimental setup, increase the number of PCR cycles (3–5 cycles at a time), up to 40 cycles.A positive control should always be included to ensure that each component is present and functional. First, ensure that all PCR components were included in the reactions.
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