Sorry basic question. I am aware primers should be described in the 5'-3' direction in the literature. When I am ordering primers I have taken from published papers, should I use the same sequence or make a reversed and complemente strand for ordering the reverse primer?
I would use the published primers but check them using primer blast to make sure that there are no typological errors in the primer sequences as published
Wilsea Maria Figueiredo Usually, yes. But it is always a good idea to analyze any published primers using the many available online tools to check out the specificity and thermal/structural profiles. At https://genome.ucsc.edu/cgi-bin/hgPcr, you can do in silico PCR on the genomes of dozens of different eukaryotes as well as on the mRNAs of human and mouse. You can also use Primer Blast at https://www.ncbi.nlm.nih.gov/tools/primer-blast/ on any sequence that you specify or ANY sequence in Genbank with an Accession number to check published primers or design novel primers. Other tools like Primer 3 at https://primer3.ut.ee/ or https://www.idtdna.com/pages/tools/oligoanalyzer will show you whether there are problems like excessive primer-primer homology or possible stable hairpin formation. For bacterial genomes, the site at http://insilico.ehu.es/PCR/index.php can also be useful.
You can obtain lots of information with just a short bit of computer time effort and this may help you avoid unpleasant surprises and lost time and effort in the lab. Another thought is that primers are usually cheap, but lost lab time is expensive, so even if you order "published" primers, you should try to design another pair to achieve the same goal and order those too. I've often found that self-designed primers perform better, especially after you gain some experience in focusing on those characteristics that affect performance in your own hands.
@Wilsea Maria Figueired- For ordering the primers we have to follow the following steps-
1) download all the sequences of specific organism from NCBI
2) do alignment using available tools
3) Identify your target region which you want to amplify
4) Find out primer sequence (published/own prepared) in aligned file. it is better to select most conserved primer sequence if you are preparing your own primer.
5) by using available online tools for primer preparation, check the parameters(GC contents, Tm, length of primers).
6) for reverse primer preparation we have to reverse and complement and follow the same procedure followed for forward primer preparation.
7) But while preparing primer order sheet we have to mention both primer strands from 5' to 3'.
Thanks
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