I need to clone ftsZ gene from Mycoplasma gallisepticum. But the gene has 2 stop codons inside of it. I was told to use Gibson assembly, but I'm not sure how.
Design primers that anneal before the stop codon when you amplify the gene.
Gibson assembly is a method of DNA assembly that enables DNA fragments to be combined in a single reaction. To remove a stop codon, the fragments of the gene should be designed such that the stop codon is replaced by a desired codon. The fragments should then be assembled using Gibson assembly, which will result in a gene without the stop codon.
Do you have the gene containing DNA fragment already on a plasmid (in E. coli)?
If yes, you can fast and easy do site directed mutagenesis PCR, this is a very efficient and useful method to quickly perform nucleotide substitutions or deletions. All you need is a PCR setup with a specific primer pair containing your sequence of interest (in your case: The sequence without a stop codon, where the nucleotides around the original stop codon should be in the center of your primer) and the DpnI enzyme.
With this primer pair you amplify the whole plasmid (which was extracted from E. coli) by PCR. Afterwards, you digest the template plasmid with digestion enzyme DpnI. Next, you transform E. coli with your digested PCR product, extract the plasmid DNA, and repeat the whole procedure with primers for your second stop codon.
You can see here, for more detailed information: https://blog.addgene.org/site-directed-mutagenesis-by-pcr
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