I am still compiling my data into a comprehensible paper, so there are no new experiments yet to report. I will only be doing lab work this semester if I run into incomplete notes on data in my lab book. If that happens, then I will need to re-run that particular experiment to complete my data set.
I was going to explain primer design on this blog, but instead I found an online resource that explains it more thoroughly than I ever could, so I have included a link to it here: http://www.cybertory.org/exercises/primerDesign/ . The lesson from Cybertory.org is one of the simplest and on-point explanations I have ever found and is a great introduction to how primers work.
I am also including a link here from Bioinformatics.org that will assist you in designing your own primers. Of course, if you would like to manually reverse-complement your DNA strands, go for it, but I prefer an online generator to reduce the chance of transposing a nucleotide and rendering my primer useless. With the Bioinformatics link, you just type in the gene sequence you are targeting, then click the function you need (reverse/complement, etc.), and your new primer sequence is immediately generated.
There are two other great resources for finding gene sequences and/or designing primers. One of them is the NBCI database, which contains an extensive listing of genomes. I have included a Youtube tutorial here (and below this post) which explains how to use the database. The home page pictured in the tutorial has changed since it was posted, but the process is still the same (the format is just slightly different). Once you find your strain of bacteria, you can simply click the "Get Primers" button and they will be provided for you, along with useful information such as the region of the gene being targeted. You can also select your parameters, as far as base pair size, by using the editing tool provided.
Another good resource is the Straininfo.net database located here. This database is particularly useful if you are using a patented strain of bacteria, are targeting the 16s rRNA gene, and are unsure of the original source strain. This was the case with my E. coli. Simply go to the link, then type your strain in the search box (for example, ATCC 4157). This will return a page that displays an overview of the strains available, and yours should be highlighted if it is in the database. After verifying that yours is available by finding the highlighted area, just navigate below the strain listing and click the "SeqRank" icon. On the bottom left of the page will be the recommended 16s rRNA sequence. Just copy and paste, edit the weird formatting so that you have a single continuous nucleotide sequence, and you are ready to analyze your gene for potential areas to target with primers.
One final word on primer design. There are other online tools available to help you design your own set that will allow you to load in genes and scan for similarites. In my case, however, I resorted to simply downloading all of the 16s rRNA sequences for the bacteria used in my study into a single Word document. Then, I looked for correlations between the genes by using the "Ctrl F" function. It was painstaking, but fun.
I hope this helps with your own primer design. See you next blog!
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