Deinococcus Aquaticus 7kb Plasmid Isolation

Introduction


Earlier this week in lab, a 1% agarose gel was run on a plasmid from Deinococcus Aquaticus which we had grown last week. This gel had shown a 14kb band but none was the 7kb band we were looking for. We hypothesized that we needed to either incubate our sample in lysozyme for a longer period of time or use a different plasmid extraction kit in order to achieve better results. This was due to our sample not becoming clear after the addition of neutralization buffer from a GeneJET Plasmid Miniprep Kit. We will run another plasmid isolation using the same kit as a control in order to test this hypothesis.

Procedure


To prepare the sample for the ThermoScientific GeneJET Plasmid Miniprep Kit, I began by labeling 2 microcentrifuge tubes "A" and "B" respectively. I then tranferred 1mL of our TGY Deinococcus Aquaticus sample into each tube, and centrifuged for a minute. I carefully discarded supernatant and repeated this process 3 times for a total of 3mL in each tube. After this, I suspended 500ul of multibuffer wash into each of my tubes and incubated for 5 minutes at room temperature. I then centrifuged for a minute and discarded supernatant. I repeated this wash once more and discarded supernatant after centrifuging. I then added 600ul of lysozyme into each tube in order to soften cell walls. These were then incubated at 37°C for 30 minutes rather than 15 minutes. After this step, I followed the ThermoScientific GeneJET Plasmid Miniprep Kit directly as the protocol required , performed a nanodrop reading on both samples, and labeled and stored samples appropriately.

Results 


After performing nanodrop reading on both of our isolated plasmids, we can compare the following results:

15 minute incubation in lysozyme (2/02/23)



30 minute incubation in lysozyme
(2/03/23)


A 1% agarose gel has not been ran on these samples yet.


Conclusion


Our more recent nanodrop readings were cleaner than our previous one from this week. As a control, we can assume this was due to longer incubation time with the lysozyme. Our next step would be to run a gel on these samples in order to confirm a 7kb band,  however, most of our sample will need to be used in order to run this gel. Therefore, we may need to amplify our samples with PCR or gel excise in order to preserve our samples. For now, an agarose gel has been prepared for future usage. 


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