7 day RNA isolations


This week we completed the 7-day desiccation and RNA isolation process for Deinococcus pimensis and Deinococcus deserti, as well as completed our side projects pre-desiccation protocol. Despite a few minor setbacks, the experimental outcomes showed promising potential.

Our findings demonstrate the RNA's capacity to survive for up to 8 days. With this side project mostly completed, the team can now redirect full attention to the reference gene project. Optimistically, the RNA-seq phase is anticipated to commence within the next couple of weeks.


Protocol:

The RNA isolation procedure remains consistent with previous protocols,

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 5 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


The procedure for cell packing and plating for desiccation is as follows:

1. Suspend cultures in 10mL of nuclease-free water

2. Take OD reading and normalize to 1

3. Centrifuge cultures (5000 RCF, 4°C, 5 min consistently)

4. Remove supernatant, resuspend in 4mL water

5. Spin down cultures, remove supernatant, resuspend in 4mL water

6. Spin down cultures, remove supernatant, resuspend in 2mL water for plating

7. Take final OD value

8. Plate 12 100μL samples onto kapton, each in a well of a 6-well plate


We started with an OD of 2.89 and 2.69, which we normalized to 0.96 and 0.98, and ending with much more culture (29ml and 27ml) then we ended up working with. Our Kapton tape patches were autoclaved then placed in the center of the wells, with the normal kapton squares being placed on top of those. We dont usually do that but as these samples were being sent away from lab we wanted to take the precaution. During this procedure the pellet had a difficult time staying together but we got through the procedure just fine.


Discussion

Complications arose during the 7-day sample RNA isolation due to mislabeling, rendering the deserti sample unusable. Conversely, the pimensis sample remained intact and demonstrated favorable results (190.7ng/uL, 2.01, 2.17). 

An overlooked 24-hour deserti sample from the previous week, which had been stored for a full week, was processed on 8/28/24. The first set of results comes from a sample plated 24 hours prior, while the second set is from the 8-day-old sample. Both were plated directly into well plate wells, diverging from the kapton plating procedure used in desiccation experiments. Generally, these samples exhibited lower yield compared to typical kapton-plated cells. Although not as good as the previous week's samples, the results remain promising if we do a cleanup before using them. 



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