S-STEM Scholars Sam Bennett's Blog

  Introduction           We started this week off with restriction enzymes. Learning about restriction enzymes is imperative to developing laboratory methods as well as learning about genetic engineering. A restricted enzyme is a protein that has been isolated from bacteria, producing DNA fragments. We started with 2 micro-liters of restricted enzymes, 10 micro-liters of our buffer, 8 micro-liters of our kanamycin DNA, and then 80 micro-liters of PCR water. The main goal was to remove pst1 from our sample's DNA.  After we had our samples combined, we incubated at 37 degrees celsius for 15 minutes, which was then followed by inactive at 80 degrees celsius for 20 minutes. We then loaded the samples into our gels and then ran the gels at 85 mA for around 30 minutes.  Once the gel had run its course, we placed the gel over the UV light and found out the kanamycin did not split how we were hoping it would.  The following day we ran an additio...

  Introduction     In the process of learning new things, sometimes science can become redundant. This week we worked on the Left Flank of our sample and went through the gel extraction process. We started with making a 1.5% gel to run our samples through. To make this we need 30 Ml of 1X TAE and 450 mg of agarose powder. While we waited for our gels to solidify, we tested our samples on the nano-drop.  From this we got:   Ng/Micro-Liters A260/A280 A260/A230 L1 245.9 2.01 2.04 L2 208.4 2.01 1.39 L3 233.9 2.03 2.07 L4 209.0 2.04 2.09     After the gels finished solidifying, we added our samples. We ran 18 micro-liters of the DNA sample, and 2 micro-liters of our blue dye.      After the gels had finished running their course, we placed them ...

 Introduction     This week was preparation leading into next week's project, ethanol precipitation. Ethanol precipitation is used for concentrating and de-salting nucleic acid (DNA or RNA). This week we were running our samples through gels and then extracting our samples once they've run through the gels. We started with a different variation of our usual 1% gel, this time we used a 1.5% gel. For this, we needed 30 mL of 1 time TAE and 450 mg of agarose powder. With the addition of an extra 5 seconds in the microwave and 150 mg of agarose powder, our gel is made. While we were running our gels,      We set the Nano-Drop to Double Stran DNA and tested the samples.  The reason there are 2 pictures is that I ended the experiment by accident....     By the time we finished on the Nano-Drop our gels had finished and it was time to place them over the UV light and extract the sample.      We measured each 2mL tube that the samples ...

 Introduction      Last week we worked with the Left Flank, as well as the Kanamycin resistant gene from e.coli, in our effort to transform Deinococcus Radiodurans. This week we worked with the Right Flank. First, we started with making our 1% gel to run the samples through. While we waited for the gel to solidify, we Nano-Dropped our Right Flank sample.      From this, we got: 280 -2.71, 260 -5.10, 230 -2.27      Finally, our gel had become as solid as we wanted. We loaded our 20 micro-liter sample (2 micro-liters of dye, 18 micro-liters of DNA) into 2 wells, and made sure to load the ladder. After we ran the gel it was time to place it over UV light and carefully remove the fragment.  This photo is from last week but added to emphasize the procedure.      After we extracted the fragments we placed the Right Flank as well as our other fragments into the -20 degrees Celsius freezer to chill overnight. The following mo...