Dear all,
has anybody some experience with using denatured ethanol in nucleic acid extraction (instead of molecular grade ethanol)? I cannot find any data/description about possible inhibitory effects of downstream PCR reactions. The denaturant could be any that is on the market (e.g. MEK/butanone).
I know pure Ethanol is of course the best solution, anyway I am curious and a bit surprised there are no (obvious) sources. I would be happy if you could share any hints/sources/experience you have!
BR,
Christian
Generally for molecular biology grade ethanol, tests are required from the manufacturers to ensure a low level of DNAse / Protease
Denaturants in denatured ethanol, such as methanol and isopropanol, can potentially have adverse effects on PCR (Polymerase Chain Reaction) if they are not completely removed during the DNA purification process. PCR is a highly sensitive molecular biology technique that can be influenced by the presence of contaminants. Here are some potential effects of contaminating denaturants on PCR:
Inhibition of DNA Polymerase: Denaturants like methanol and isopropanol can inhibit the activity of DNA polymerases, the enzymes responsible for DNA amplification in PCR. Even small amounts of these substances can interfere with the enzyme's function, leading to reduced PCR efficiency or complete failure of the reaction.
Reduced Amplification Efficiency: Contaminants in denatured ethanol can lead to reduced amplification efficiency by interfering with the binding of DNA primers to the template DNA or by affecting the stability of DNA strands during the temperature cycles of PCR. This can result in lower yields of the PCR product.
Altered Melting Temperatures (Tm): The presence of contaminants can alter the melting temperature (Tm) of the DNA template, which is the temperature at which the DNA strands separate during PCR. Changes in Tm can affect the specificity of primer binding and result in non-specific amplification.
Increased Non-Specific Amplification: Denaturants can promote non-specific binding between primers and DNA, leading to the amplification of unintended DNA fragments. This can complicate the interpretation of PCR results and decrease the specificity of the assay.
To minimize the impact of denaturants on PCR, it is essential to use high-quality DNA purification methods that effectively remove any residual denaturants. Common DNA purification techniques, such as phenol-chloroform extraction, ethanol precipitation, or commercial DNA purification kits, are designed to eliminate contaminants and ensure the purity of the DNA template used in PCR. Additionally, using molecular biology-grade reagents, including ethanol and other chemicals, can reduce the risk of contamination in your PCR reactions.
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