Growth curves and Freeze backs

This week was mostly full of inoculations. We started the week both with new growth plates as well as a flask for a growth curve, and were able to help out Evans group with some inoculations later in the day as well. We both made some media and poured some plates to make sure we have enough to finish the semester.


We wanted to get a growth curve to make sure we were growing for the optimal amount of time. Bacterial populations grow in a pattern that we can see in a graph and this pattern includes special phases: lag, exponential, stationary and death. During the lag phase, bacteria prepare for reproduction and change to new ecological conditions by synthesizing enzymes. In the exponential phase, cells divide quickly and the population doubles consistently at set intervals. In the stationary phase, growth rates drop as nutrients run low and waste builds up. When the population starts to decline because of resource depletion, and the buildup of toxic metabolites, it is during this period that the death phase finally occurs.



Unfortunately with the way deserti has been clumping lately, we were only able to get the first few hours of the growth curve before it clumped into a little ball inside the flask. 


From our previous freeze backs we had A, B and C as we needed three biologicals. However the recent trends in our RNA isolations have been that out of the three we always end up with one that is worse than the others making it so that we can't use the run. Because of this we will be switching to 5 biologicals. Because of this we took freeze back tube A and plated it. After 48 hours of growth we inoculated 5 separate colonies onto new plates making biologicals A-E. Later in the week we used those to inoculate 5 plates in order to make a freeze back, and 5 flasks for RNA isolation the next week.

 

With all of the inoculations came a number of gram stains. As time has gone on we have noticed a trend with the bacteria looking slightly smaller and more circular as time goes on and the flasks getting larger and larger clumps until the last few growths have had a single large pellet. The media in the flask still has a good amount of bacterial growth, but the clumps contain a majority of growth. 



As we did less protocols than usual this week, we were able to go over the sequencing kit again and divide up the sections so we have a equal workload. Most of the solutions will be prepared ahead of time so we just need to pipette in the mixed final volume. Madison will be doing the reverse transcription and strand switching step while will do the PCR and adaptor addition steps. Chad will be doing the cell preparation and loading step. We also compared our current procedure to one that includes barcodes, but the two procedures are actually the same except for one solution added in a single step. Step four of the full length transcripts in our current procedure has 5ul sample, 1.5ul cDNA primer, 18.5ul nuclease free water and 25ul long amp master mix. The barcode procedure has 5ul sample, 0.75ul barcode, 6.75ul nuclease free water and 12.5il long amp master mix.

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