RNA Isolation and Planning

 Like usual we started this week with RNA extraction, as well as inoculating for and planning the upcoming weeks. We made new media and inoculated from freeze back to prep for pre-desiccation next week. Our RNA sample was from a four day desiccation period, and we went from 500ul to 250ul of rehydration buffer to make up for a limited supply of the buffer this week. The samples also sat in the rocker for 10 extra minutes to properly rehydrate with the limited amount of buffer.


RNA Isolation:

1. Resuspend a fresh or frozen pellet in 800μl RNA lysis buffer and transfer the mixture to a ZR bead bashing lysis tube

2. Secure the tube on a bead beater with a 2ml tube hold assembly and process. Repeat 2 cycles of 1 minute on and 2 minutes on ice

3. Centrifuge the tube for one minute to pellet debris

4. Transfer up to 400μl of the cleared supernatant into a Zymo-Spin IICG column in a collection tube and centrifuge for one minute. Retain the flow through

5. Add an equal volume of ethanol (95-100%) and mix thoroughly

6. Transfer the mixture into a Zymo-spin IICR column in a collection tube and centrifuge for one minute. Discard the flow through

7. Add 400μl RNA wash buffer to the column, centrifuge for one minute, and discard the flow through

8. Prepare the DNase 1 reaction mix (75μl DNase buffer and 5μl DNase per tube), add 80μl directly to the column matrix, and incubate at room temperature (20-30°C) for 15 minutes before continuing purification

9. Add 400μl RNA prep buffer to the column and centrifuge for one minute. Discard the flow through

10. Add 700μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

11. Add 400μl RNA wash buffer to the column and centrifuge for one minute. Discard the flow through

12. Add 400μl RNA wash buffer to the column and centrifuge for two minutes. Discard the flow through

13. Transfer the column to a nuclease-free tube, add 50μl DNase/RNase-free water directly to the column matrix, and centrifuge


ng/ul

a260/280

260/230

A

69.8

2.030.47

B

41.72.021.82

C

95.62.051.32



Sample B was lower in yield and samples A and C were lower in cleanliness, but overall the samples were all good and fully RNA. With our procedure mostly worked out we have decided to continue working to obtain a clean sample for sequencing. However we will not do a clean-up on these samples and instead get another set. This is when we spent our time making the plan for this and next week, as well as made our media and inoculated our plates. 

The Plan is as follows: allow the A, B and  C plates inoculated this week to grow over the weekend and inoculate flasks A-F, with D, E and F being controls (A and D, B and E, C and F). Then those grow for two days and we do a pre-desiccation procedure including gram staining, creating freebacks A B and C, and getting some CFU spot plates. Then post pre-desiccation procedure they will desiccate for 5 days then we will perform RNA isolation.  

Comments

Post a Comment

Popular posts from this blog

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...
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...
Read more

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 ribof...
Read more