I just want to know a question in terms of the result I got after PCR. The final PCR products contain my template DNA, amplified DNA, and dNTPs. How can I distinguish the parental DNA and amplified DNA? I just introduced a point mutation and they should be the same size.
You typically only use a few nanograms of template DNA, which should not be seeable on an agarose gel. Therefore, if you see a crisp band, it can only be the amplified DNA.
dNTPs sometimes form a very subtle cloud or smear at the bottom of the gel, lower than 100nt compared to a DNA ladder. But you absolutely can't confuse those with an amplified DNA band. See attached file for an example. In this image, you can even see some primer dimerization (where the primers anneal to each other and amplify each other).
The concentration of the template must be low enough when you see band it will confirm that it is the one amplified to contain the SNP (you can compare by running a well containing the same amount of DNA used as template to make sure it is not visible). The parental DNA is likely methylated therefore dpnI digestion after the PCR will remove the template while leaving the product intact. If the snp you are introducing happens to remove a restriction site then you can use that restriction enzyme to remove the fragment. That being said if you are using the amplified fragment for downstream application (like cloning in a plasmid) that concentration of the amplified fragment will be higher than the template thus you will not have a problem detecting correct clones.
You typically only use a few nanograms of template DNA, which should not be seeable on an agarose gel. Therefore, if you see a crisp band, it can only be the amplified DNA.
dNTPs sometimes form a very subtle cloud or smear at the bottom of the gel, lower than 100nt compared to a DNA ladder. But you absolutely can't confuse those with an amplified DNA band. See attached file for an example. In this image, you can even see some primer dimerization (where the primers anneal to each other and amplify each other).
Add more dye to your amplified DNA, invert your gel image, Run gel band analysis using mega 7 software and the difference will be glaring.
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