Lysing Aquaticus and Trying to Get a Plasmid

 

    At the end of last week, I had tried (and failed) to get a plasmid from Aquaticus. In addition, while going over the lysing procedures over the week I made some adjustments and vastly increased the genomic DNA yield. 


    Starting on Monday, I want through the lysing procedures. To be honest, not even thinking about it I vortexed the pellet in the special multiwash before letting it sit for five minutes, and vortexed it again before putting it into the lyse. While it wasn't in the protocols I knew it would help break up the pellet and let the multiwash/lyse reach more cells than the pellet. When I realized what I had done, I was worried I had messed it up. Instead we saw the highest DNA yield yet. We  even re-nanodropped it, and checked the nanodrop against other samples to make sure it wasn't broken. 


  

    After nanodropping, I did the zippy plasmid extraction kit. However, after running the gel we saw nothing but the ladder, not even a smear or something stuck in the well. The ladder was also not very good, and I think the gel itself had some issues, however that wouldn't make the plasmid disappear. 



the bands should have been to the left of the ladder



    At the end of last week Lani and Ryan had gotten the aquaticus plasmid, so we knew it was there, we just weren't getting it. Our protocols were slightly different, and we were using different Lysozyme. Beyond the lysozyme the differences were mainly time sat in the waterbath or amount of lysozyme used (500 vs 600 microliters). 

    We began to think that the difference in lysosyme was the difference in why they had gotten the plasmid and I hadn't, and I was planning on running two experiments, both with my protocols. One would have my lysosyme and one would have theirs, and after I would run the zippy plasmid extraction kit and try to see the plasmids on a gel. We also talked about running more trials after the lysosyme one to try to optimize it as much as possible. 

    When talking I learned another difference: After taking their lysed bacteria out of the waterbath they moved into the plasmid extraction as soon as possible, while I normally waited about 10-20 minutes more getting everything together. I think this may be a reason I haven't gotten the plasmid. I think the lysosyme may be effecting the DNA, but I'm not sure how that would be possible. To see if it has an effect, I ran through the new protocols (including vortexing), and after taking the lysosyme out of the water bath, immediately nanodropping and moving onto plasmid extraction. This would allow me to prove not only the vortexing, but also see if the amount of time in the lysosyme effected the plasmid. 



Latest Lyse Protocols-     

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

vortex until pellet is completely mixed into wash

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

vortex until pellet is completely mixed

Place in water for 5 minutes


 
nanodrop as soon as possible and if trying for plasmid, move onto zippy plasmid extraction protocols asap, starting at step 2.


    
    After performing the lyse protocols, I nanodropped to confirm that the vortexing worked. The 700 sample started with a larger amount of bacteria, and both results are at least three times as high as results without the vortexing. 



545 ng/μl


    After nanodropping I immediately moved on to the zippy plasmid extraction protocol, however I didn't have time to run a gel, so ill be doing that monday. If it doesn't show a plasmid, I'll do the experiments with the two different lysosymes. I also made a new sample of Aquaticus that is incubating, as our old stock was starting to get old. 

    All in all I feel like this week was very productive, and while we had some success and some failures, I learned a lot and have a lot planned moving forward. 


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