S-STEM Scholar Alex Hugenberg-Cox Blog

  This week I continued working on my current project: replacing a section of  Deinococcus Radiodurans's DNA with  tetracycline resistance using restriction enzyme ligation. While I was away the project was continued through the phosphorylation and digestion steps, leaving us to finish the final Ligase procedure to finish the project.  T4 DNA Ligase steps/procedure:   We started by setting up the reaction in a micro-centrifuge tube that would be kept on ice; and added all the components      17  μl of various combined fragments        2  μl of T4 DNA ligase buffer       1  μl of T4 DNA Ligase T hen we gently mix the reaction with a mix of pipetting and briefly microfugeing    Because we wanted cohesive (sticky) ends we incubated at room temperature for 10 minutes.   After that we kept it at 65 degrees Celsius for 10 minutes   We then kept it on ice until 1-5 microliters could be added to 50 microliters of cells.  After we finished the ligaseing we did both normal and long

  Magnetic fields are created when electrons spin. Normally electrons are paired, and if they spin in opposite directions from magnetic fields cancel out. However in free radicals the electron sometimes are unpaired or a pair electrons spin in the same direction, in this case the magnetic fields are not cancelled out. Most enzymes are not affected by these magnetic fields, however some are. This experiment is to determine the amount of effect of a magnetic field on the enzyme known as superoxide dismutase aka SOD.   SOD is a catalyst that catalyzes the conversion of superoxide free radicals into O2 and H2O2. This experiment was to study the influence of magnetic fields on SOD activity, to see how it would affect biological processes. They tested this by exposing SOD to EMF for various amounts of time then measuring its activity by measuring its reaction to oxygen. To conduct the experiment, the SOD enzyme was combined with Na2CO3, methionine, NBT, EDTA, PBS, dH20 and riboflavin Sol

  Linear Plasmids, Restriction Enzyme Ligation, and Plasmid Extractions   The project I’m currently working on is linear plasmids. We’re trying to take a plasmid from E.coli that contains a gene called tet that codes for tetracycline (an antibiotic) resistance, and remove D.rad’s (Deinococcus Radiodurans) lux.s gene before incorporating tet into the spot in D.rads genes where lux used to be.   On Monday we decided to separate into two groups and try two different methods of removing the lux and replacing it with tet. While the other group will be doing the original overlap PCR, our group will be trying restriction enzyme ligation. Both groups will be performing the same basics, but going about the process in different ways. In the end we’re just trying to find which process is more reliable and efficient.   Before we could start doing restriction enzyme ligation, we needed to create more plasmids, as the various experiments over the week have depleted our supply. Using New