Aquaticus Lyse

 

    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 dissolve. 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 tris HCl, and 85 ml of H2O.

    

    Making the lyse solutions was much easier. I had a Tris HCl solution that i brought the pH to 8.3, and another Tris HCl solution that I dosed with lysozyme and brought the pH to 8.2. 

    After that making the solutions was talking through making the protocols with Dr Tuohy-

Protocol for the aquaticus lysozyme 

Remove 3 ml of aquaticus sample from large flask (3 per tube you will be doing)

Do not pipette directly out of large flask that contains mass sample. 

Use 5ml disposable pipettes. 

Use 1.5 ml or 2 ml centrifuge tubes with the pointed ends. 

Heat waterbath to 37.5 degrees celsius. 

Step 1. 

transfer 1 ml (1000 microliters) of sample into tube

centrifuge pellet (1 min)

discard supernatent

repeat step 1 three times, 3 ml total

Step 2.

add 500 microliters of multiwash

let sit for 5 minutes

spin to pellet

discard supernatent

repeat step 2 twice

Step 3. 

add 450 microliters Tris HCL

add 50 microliters Tris HCL Lysozyme

Place in water for 5 minutes

nanodrop

    Before the first test, I nanodropped our sample of cells. 

 

    I then did our first test, going through the protocols and nanodroping at the end. 

    The next day I did a variation, going through the protocols with 4 tubes (A, B, C, and D) and keeping them in the water bath for different amounts of time. (5 minutes, 10 minutes, 15 minutes and 20 minutes respectively. I once again started with nanodroping the sample, and nanodroped the results at the end. 

beginning sample nanodrop

timed tests nanodrop (A, B, C, D and A again)

    I'm unsure about the results of the A tube, just because the numbers are so much higher than any we've done, but its also shown that while 10 to 15 minutes doesn't change much, after that the numbers do start to drop. 

    Another part of our project is trying to extract a plasmid from aquaticus, and I spent the next day doing that. However to be completely honest, I completely followed the zippy plasmid extraction protocol, including the lyseing part. As one may have guessed, that did not turn out very well, as to lyse those cells we have to go through an entire process. However this did not occur to me until after i had done the extraction and all i could see was the ladder. I'll do the plasmid exraction next week after lysing new bacteria, and while using by brain cells. :)

ladder, and sample was ran to the right of it

Kikuchi, M., Kitayama, S., Sjarief, S. H., & Watanabe, H. (1994). Plasmids in Several Strains Deinococcus radiodurans [Review of Plasmids in Several Strains Deinococcus radiodurans]. Radiation Research, 139(1), 123–125. JSTOR. https://www.jstore.org/stable/3578752