Multimedia Tests and RNA isolation

     We started this week off with the results of the inoculated multimedia test last week. We made and inoculated TSA, NB, R2A, TGY, TGY salt at 1% 2% and 3%, Mueller Hinton, EMB, Mannitol, Maconcy, PEA and LB media. However Deinococcus sonorensis only grew on TGY, R2A and slightly on NB. The mannitol plate had slight growth but after letting it grow an additional two days and it was eventually decided that the initial few colonies did not count for actual growth on the media. 

    In addition to the media from last week, we have decided to make altered TGY and R2A media to see how the bacteria would react to different levels of nutrients. A 1/2 TGY, regular TGY and x2 TGY and a 1/2 R2A, regular R2A and x2 R2A. We did have to remake some after issues with the autoclave ruined about half of the altered media, but we just remade it.

    The biggest thing about the initial R2A and TGY plates in the multimedia test wasn't the fact that it grew on the R2A but the fact that when it did it lost the hard plaque structure that made it so difficult to work with. When inoculating on to other plates the loop picked up the TGY grown bacteria in clumps that retained their shape and make it difficult to spread or thin. However the R2A grown bacteria was easily grabbed and spread, acting and looking like a normal bacteria. We need to do further testing including gram staining and growing in broth, but this is looking like a very interesting phenomena that others are going look into bioinformatically while we continue to characterize.

NB, R2A and TGY plates


    I also spent a day going over the AMC cycle and our past work with the group, teaching them about the pathway. We performed another RNA isolation, but only on one test/control pair was the other two flasks did not have high enough OD values after the normal incubation period. The Exposure was done as normal. 70 mJ/cm^2 for two minutes, with the E.coli being grown that morning then normalized, exposed and diluted. After the 30 minute recovery at 30°C we moved on to RNA isolation. 

Initial OD values

A- 0.16

B- 1.05

C- 0.09

    The RNA isolation itself was done with only one round of bead beating, however the results this time has some organic contamination and a high ng/μl, but the A260/A230 values were both only 0.83. This RNA isolation was supervised to see if there were any technical errors and we were deemed clear, so the issue at this point is somewhere in the tools. We will be reviewing the kit and making sure that everything is good as well as taking extra care with the pipettes. 


Comments

Post a Comment

Popular posts from this blog

Summary of 'the effect of magnetic field on the activity of superoxide dismutase'

  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
Read more

Aquaticus Lyse

Image
      In the last blog I mentioned moving onto another project with Kieran, trying to replicate a D.rad lyse onto aquaticus. Before I could start with the procedures I had to make the solutions and create the protocols. It was a lot of dilutions and changing the pH of things, but I also had to make an EDTA solution.       To make the multibuffer I would need, I started by making 100 ml of 50 mM sucrose, and 100 ml of 10 mM tris-HCL. After making those two solutions, I made 100 ml of .5 M, pH 8.0 EDTA. While it was simple enough to combine the ingredients (18.61 g disodium EDTA- 2 H2O and 80 ml water), I then had to change the pH. The original pH was around 3.4, so it took a while, dosing it with HCl to raise the pH and cause the solids to dissolv e. After the EDTA was diluting the 10% Triton x-100 to .1%, which was done when creating the multibuffer. I ended up making 100 ml of multibuffer, with 8 ml of the EDTA solutiom, 5 ml of the sucrose, 1 ml of the 10x Triton x-100, 1 ml of the
Read more

Linear Plasmids, Restriction Enzyme Ligation and Plasmid Extractions

Image
  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
Read more