I would like to insert my cDNA of interest, Xdh, in a plasmidic vector in order to transect my cells. I have already designed a pair of primers for the adding of the restriction sites in my cDNA. i don't have cDNA or mRNA of my gene so I have to extract fresh from a tissue sample. My doubt is this: can I extract directly DNA and use it as a template for the amplification with the primers leading the RE sites avoiding to design another pair of primers for the mRNA?
The question is if I have to start from a mRNA pool and make a retro-transcription in cDNA, and use this as a template or I can extract directly DNA?
Thank you so much
If your DNA source is prokaryotic (bacterial) you can go ahead as planned. If your source is eukaryotic, then you may have trouble, as eukaryotes have introns in their genomic DNA. perhaps you are fortunate and your gene of interest has only a single intron. you will need to check this in the gene database. If there is an intron, you will need to isolate RNA from the cells and reverse transcribe it to cDNA to use as template for PCR. good luck!
Joseph, you are absolutely right; to start from a mRNA means that you have the entire coding region of the gene, and not introns. For the RT-PCR of my mRNA do I need a specific pair of primers to convert only the mRNA of interest or I can retrotranscribe all the mRNA. In case I need a pair of primers, do these primers fw and rv need to be designed for the both ends of my mRNA, or are casual primers?
Thank you so much
Typically reverse transcription is done for the whole pool of mRNA and then that is used as template. Usually oligo dT (annealing to the mRNA's poly-A tail) is used as the primer for reverse transcription, followed by the gene-specific primers of interest.
If you have a long 3' UTR and you only want the coding region it may be more efficient to use a gene specific RT primer. This way your RT reaction won't have to run the length of the UTR. If you use oligo dT I would sugest that the last nucleotide on your primer will be V (that is anything but T) this way your primer would only bind to the beginning of the polyA tail.
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