cDNA synthesis and RNA Gel

     This week we ran our RNA samples from last week, so i got to make and run a RNA gel for the first time. 

    Monday Deja transformed a small amount of our T1 RNA sample into cDNA using a new kit. Our usual kit was gone, so in the mean time we tried a small amount of our sample with a new kit that had different steps to see if it would work. The only way to see if it had worked was to run qPCR, so that's what we did Tuesday. Unfortunately it did not work, so we would need to wait for our usual kit to get it, but we still had the rest of the samples. 

    Wednesday I taught Lizzie and Iditi, to people in the lab who had never done the RNA isolation procedure, how to do it. It was good for me to sit and run through all the steps, and explain each one and make sure it was being done correctly and precisely. It helped me gain a better understanding of the procedure, and what different steps did. 

    Thursday was mainly doing a few things to help Lani out with her upcoming poster, like doing graham stains or making gels. Friday was when I finally ran the RNA gel. 

    I did mess up a few times in the beginning. At first I made the gel itself without any midori green, so I had to remake that. I also did the dilutions like normal, trying to get 10μl of sample, expecting to add 2μl of dye. However after diluting the samples I realized that the RNA gel dye was 2X, not 6X like normal dye. In the end it was fine, with maybe a microliter or so not fitting in the wells due to the large amount. And at the very end, on my way back to look at the gel... I dropped it. We pieced it back together, and the gel turned out fine, but it was a good lesson to be careful and always plan ahead. 




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