Primer+Specifics

How to resuspend and store primers: http://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded-test/2011/03/16/dna-oligonucleotide-resuspension-and-storage

Procedure for Specific Molar Solutions To make stock 100 Micromolar solutions: To make 10 Micromolar solutions:
 * Spin down both primers to ensure that no oligo is dislodged from the tubes during shipping.
 * Add 343 microliters of mucleas-free water to ITS1 (mass 34.3 nanomole). Add 403 microliters of nucleas-free water to ITS4 (mass 40.3 nanomole).
 * Spin down both solutions and vortex.
 * Take 10 microliters of each of the stock solutions. Add 90 microliters of nucleas-free water to each of them.
 * Result: Two 100-microliter primer solution at 10 micromolar concentration.

__Objective__ Using the knowledge of our primers, try to find the exact number of base pairs that we are amplifying and subsequently sequencing. __Where to Start?__ In order to find the area of the lichen DNA for amplification, we used the primer information found by White et al from Duke University in 1990 for ITS 1 and 4. We used the knowledge that primer ITS 1 (51TCCGTAGGTGAACCTGCGG31) will bind to the beginning of the sequence and isolate “down stream” on the DNA. ITS 4 (51TCCTCCGCTTATTGATATGC31) provides the termination of the sequence because it attaches to the other strand of DNA than the ITS 1 and works the opposite way, highlighting a specific region. __What is ITS?__ ITS stands for Internal Transcribed Spacer, which is a region within all lichen species, and usually amplified to highlight differences between lichens within the species. The ITS region has a large level of genetic diversity so it makes it perfect to understand and barcode the different species with the ITS region of the DNA __Why?__ The obvious benefit of highlighting and isolating a specific region is that it allows us to amplify and analyze only a couple hundred base pairs of DNA instead of the millions of base pairs of the entire DNA. __How?__ In order to find the exact number, we blasted the first primer. When we found the

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