In a case, one ORF with a TAA stopcodon at its 3’ end. Can I modify the base sequence (eg instead of TAA, I want to change as TTA to modify the stopcodon by incorporating the mutated base sequence in the 3’ end of reverse primer. Is there any possibility of changes in the same stopcodon during protein expression? . Is there any rule to make such modifications?. Usually in protein tagging (N terminal tagging with GFP) we are selecting sequences just before stopcodon to tag with GFP… I know that 3’ end of reverse primer is also highly specific/sensitive to PCR. Please give me directions
Yes you can modify the sequence by adding a base change in the primer. However, I wouldn't change this base at the 3'prime as this for sure gives issues with your PCR. I have done this before and i always design a 25-30bp primer with the mutation in the middle (sort site directed mutagenesis). Always does the trick for me. Alternatively, you can design an a-specific 5'prime overhang with the new codon....that also works. Good luck!
Yes it is possible. It is better put the change in the middle of a 25-30 bp primer. In same case I changed 2 bp together in the same primer . Good luck
Yes, it is possible. Try to make the 3' end of the new primer (with modified TAA codon) specific for 18-20 bases. What will you add after that makes no difference. I have good experience with primes with more than 40 nucleotides at 5' that were completely different from the matrix.
As everyone has said, yes, you can certainly introduce a mutation into the stop codon during PCR by changing the sequence of your reverse primer. My question is, how do you intend to go from that to protein expression? If you're using a coupled transcription-translation system or T/A cloning, you're probably already set. Conventional cloning? Make sure your primers contain whatever restriction sites you require. You can also introduce point mutations - including in the stop codon - directly into your expression vector using a QuikChange II SIte Directed Mutagenesis Kit (Agilent). You can do the same thing more cheaply by buying the kit components separately. You will need to design two primers for use with this technique, but instructions for how to do this are provided with the kit.
Yes. We often change the stop codon to another amino acid so that our constructs will read through when we clone this into our expression vectors with various tags. The 5' end of our reverse primer has the recombinase site for cloning or a restriction site for cloning, then the stop codon is changed to another amino acid, and then we continue this primer so that it includes ~30 nucleotides that are complementary to the gene we want to amplify. We use a high fidelity polymerase like Phusion or Platinum Taq. So we don't use the Quick Change Site Directed Mutagenesis Kit when we change the stop codon because we can just incorporate the change in our reverse primer and clone it in (although the Quick Change kit is an option as well and also would do the job).
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