I want to copy a target gene from cDNA into a plasmid. The primers were designed according to the CDS sequence from NCBI. But when I performed PCR reactions I could not get any target bands. So the CDS sequence was synthesized into my plasmid vector. When used the plasmid as template and the above primers to run PCR, target bands were quite clear which means the primers can work. I know the gene copy number in plasmid must be much higher than in cDNA. So I increased the amount of cDNA template and cycle numbers (from 35 to 45 cycles), no target bands showed. Could anyone tell me what the problem might be. Is that possible that the CDS sequence in my cells has changed? If yes, is there any other ways to get the CDS sequence except artificial synthesis.
Depends how divergent the sequence of that gene in your strain is from the reference genome. The simplest approach would be to design primers that amplify gDNA across the first and last exons which you can then send for sequencing and use that information to design new primers for amplifying the CDS. Alternatively do a partial digest of gDNA with a common cutting restriction enzyme and ligate at a low dilution to circularize before doing a PCR across the ligation site using primers designed to bind to highly conserved regions/domains of the gene. You can then send that PCR product for sequencing.
Are you sure that your gene of interest is expressed in the cells you are analyzing?
Hi, Robert. I designed new primers including the whole CDS region and then ran PCR using cDNA as the template. This time I got the band on the gel. After sequencing the PCR product, the DNA sequence of CDS was the same as the reference genome. But when I used the previous primers which were just from the start codon to the stop codon, I could not get any band. So what might be the reason?
What I am trying to do is to copy the CDS of my target gene into a plasmid. So does it matter if I copy more sequence longer than CDS into the vector when I use the plasmid to do over-expression?
Thank you very much. Robert Adolf Brinzer
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