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!

  • $\begingroup$ Welcome to Biology.SE! Please take the tour and then go through the help pages starting with How to Ask questions effectively on this site and edit your question accordingly. In particular, your question should show evidence of prior research and some familiarity with the relevant area of biology. Note that it is extremely unlikely that you have replaced the stop codon with a protein. ——— Please do some research on your own and then, informed by what you have learned, ask any questions you still have (ideally with references to reliable sources). Thanks! 😊 $\endgroup$
    – tyersome
    Apr 15, 2020 at 17:39
  • $\begingroup$ I have found that when learning about a new area starting with a relatively accessible and reliable source like Khan Academy is very helpful. Wikipedia is also generally a good starting point and you can then check their references. Online platforms called MOOCs offer free (or very low cost) courses on a wide variety of subjects — two I am familiar with are Coursera and edX. Finally, textbooks with a good level of detail are also freely available online e.g. from NCBI. $\endgroup$
    – tyersome
    Apr 15, 2020 at 17:40

1 Answer 1


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, 2020 at 9:18

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.