This week I will cover the basics of DNA purification and quantification. Once we have amplified the target DNA using PCR, we need to purify the product (by removing primers, unincorporated nucleotides, polymerase, and salts) and quantify it in order to get a general idea of how much DNA is in the sample; if there is not enough DNA or too much DNA sequencing may fail. I used a commercial kit (Promega Wizard® SV Gel and PCR Clean-Up System) to purify our samples. First, a membrane binding solution is mixed with the PCR product and the mixture is pipetted into a spin column containing a silica membrane placed inside a collection tube; this assembly is then centrifuged in order to bind the DNA to the membrane (while everything else flows through into the collection tube). An ethanol-based wash solution is then added and pulled through the membrane by centrifuging in order to clean the filter. Once the sample is bound to the membrane and washed of contaminants, the spin column is placed into a fresh collection tube, and DNA is eluted by adding nuclease-free H20 to the filter, and centrifuging the assembly. This purified sample is then quantified using gel electrophoresis that I mentioned previously (see my previous blog post week-6.html). With this round of gel electrophoresis however, the sample is compared to a standard called Low Mass Ladder (which contains a mixture of fragments of DNA of known size and quantity) in order to estimate the quantity of DNA contained within the sample. Once the sample is purified and quantified, it is ready to send off for sequencing. The process of sequencing will be the focus of next week’s blog entry. (Photo courtesy of Bioneer Pacific)
With only two weeks left in the program, we have officially run out of time for our usual weekly extracurricular activities. Focus has now shifted to compiling the data and preparing to present it to various audiences. We are all ready to see our hard work turn into something tangible, and the excitement around campus is palpable (punctuated with healthy doses of anxiety).
My name is Becky, and I have been local to the Coos Bay and Charleston area for two years with my boyfriend David, and my dog Mojo. I moved to this area to complete my associate's degree at Southwestern Oregon Community College in anticipation of transferring to a four-year university. I now attend online at Oregon State University and I am planning a move to Bend, OR in August to continue at the OSU Cascades campus in order to finish my bachelor's degree in either natural resources or biology.