If I have a mutated colony containing the fusion protein, mCherry instead of the stop codon TAA, and an unmutated colony which does not contain the protein. Why will the PCR products of the two colonies have the same size?

I thought that since the mutated colony contains the protein instead of the stop codon, this would cause this product to be larger in size than the unmutated product. However, from experimental data it shows that both products are of same size. Could someone please explain why this is?

Thank you!

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    – tyersome
    Apr 15 '20 at 17:39
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    – tyersome
    Apr 15 '20 at 17:40

If mCherry was successfully cloned replacing the stop codon, you would indeed expect a fusion protein that is about 27 kDa larger than the original protein. However, with a PCR you are not testing for the full length protein, but rather for a part of the target gene sequence.

The length of the PCR product very much depends on the position of your primers: If both primers are targeted at your unmutated sequence, the product size will not change by adding mCherry behind the targeted sequence. The product size of the PCR would only change, if one primer is located before or in your unmutated protein and the other one behind mCherry. This would probably result in a very large PCR product and hinder the PCR reaction from being effective.

To check for correct integration of mCherry, you would need to design a primer pair with the forward primer in your unmutated protein and the reverse primer in mCherry. This should result in a product in the fusion protein colonies, but not in the unmutated ones.

Alternatively, you could sequence your products.

  • $\begingroup$ Whoever gave a -1 to my answer, could you please explain why? $\endgroup$
    – DaRealHonk
    Apr 30 '20 at 9:18

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