2
$\begingroup$

Does adding the overhangs to gBlocks for NEB Hifi using PCR pose a big problem? The IDT website suggests that one should design the gBlocks with the overhangs. However, if I did not do this, will it be a problem?

Edit: I intent to use Q5 High-Fidelity Polymerase and the gBlocks range from 500-2200 bp in size. It's not necessary for the exact sequence to be present.

$\endgroup$
1
  • $\begingroup$ Welcome to Biology.SE. Please edit your question to provide more details such as: the error rate of the polymerase you intend to use, the size of the gBlock, and how critical it is to you that the exact sequence be present for your application. You may also benefit from taking the tour and consulting the help center starting with How to Ask. This will familiarize you with how this site works and what the expectations are for questions. Thanks. $\endgroup$
    – tyersome
    Jul 5 at 18:15
3
$\begingroup$

You could add the overhangs with PCR, then gel purify or PCR purify. I don't think it will pose a big problem as I've done it before and all of the colonies I screened were correct.

$\endgroup$
2
  • $\begingroup$ Welcome to Biology.SE. We're looking for long answers that provide some explanation and context. Don't just give a one-line answer; explain why your answer is right, ideally with citations. In particular, you are ignoring a potentially significant problem with this approach — the error rate of the DNA polymerase being used. I've posted a comment to the OP asking them to give us more detail, but please also edit your answer to be more complete. Please see the tour and help center starting with How to Answer questions effectively for details about site expectations. Thanks! $\endgroup$
    – tyersome
    Jul 5 at 18:19
  • $\begingroup$ Error rate here of Q5 polymerase is 5.3 x 10^-7 [1]. One would have to be remarkably unlucky for error to propagate into your final clone. [1] Paulo Cseri Ricardo, Elaine Françoso & Maria Cristina Arias (2020) Fidelity of DNA polymerases in the detection of intraindividual variation of mitochondrial DNA, Mitochondrial DNA Part B, 5:1, 108-112, DOI: 10.1080/23802359.2019.1697188 $\endgroup$
    – JEJS
    Oct 27 at 13:04

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.