I have got a gene that I want to cut out of its present vector and ligate it into another vector, but the stop codon is the problem. Any suggestions?
PCR with modified primers is a common way there. Make sure you're omitting the stop codon with the primers, but are introducing LINKER - space between last protein's AA and 1st fusion's AA.
PCR with modified primers is a common way there. Make sure you're omitting the stop codon with the primers, but are introducing LINKER - space between last protein's AA and 1st fusion's AA.
I agree with Jose ! Directed mutagenesis is the easier way to do it...
I remember that I did use successfully both primer-mediated mutagenesis with recombinant pfu DNA polymerase and QuikChange Site-Directed Mutagenesis Kits from Stratagene 10 yrs ago....
I have been using PCR based approach. So omit the stop codon part by designing a primer complementary to your end which skips those three bases of stop codon.
Say like you go for GFP fusion and your GFP is in C term n hence your gene's stop codon has to be removed, your reverse primer with restriction site should omit the stop codon and generally vectors like eGFP N1 or C1 have additional sequences in between the fusion which are in frame and code for not very imp. amino acids acting like linkers so you may not want to add sequences there. Ya your overhangs can have vector specific flanking bases for better complementarity and ligation.
So a forward primer will have Overhang-RE site-gene Seq (may or may not be kozak) 5'-3'
reverse with gene seq (without stop codon)-RE site-overhang from vector backbone) 3'-5'
PCR or simply selecting a plasmid with an N terminal tag have worked best for me in the past. PCR is the best choice. The N terminal tag isn't a good idea if you're dealing with DNA which is not optimized for your expression system. For example, a mammalian sequence in E. coli will often result in protein fragments ending at each rare codon. On the other hand, if your gene is properly designed then you should have no troubles with a N terminal tag.
PCR with primers that include restriction sites and several nucleotides overhanging. And remember to exclude the stop codon from the reverse primer!!
Your stop codon is either TAG, TGA, or TAA, so for your 3' primer, you would use ~18 nucleotides from your gene and in the primer, instead of TGA, you can change this to the codon for some other amino acid, like leucine CUA, for example, and then the amino acids for your restriction site. The 18 nucleotides will be complementary and bind to your gene in the PCR step, and the rest, the leucine codon and the restriction site will hang off. Since this primes the DNA amplification, it will get extended in the first round of PCR, and then amplified in subsequent rounds. In this way you will have incorporated both the restriction site to clone into your next vector as well as changed the stop codon in one PCR step using a high fidelity polymerase. This is less expensive than site-directed mutagenesis and the 2 primers you order can also be used to screen the colonies in the next step after you ligate your PCR product into your expression vector. The restriction enzymes you choose in the multiple cloning site, will determine how many nucleotides at the end of your gene will be needed for effective cutting, but by replacing the stop codon with the nucleotides for leucine, you will always have a minimum of three.
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