Inoculation of Deinococcus Radiodurans for Future 7kb Plasmid Research

Introduction


Last semester in lab, a plasmid isolation was done on Deinococcus Aquaticus. when this plasmid was run on a gel, a 7kb strand was visible.  No information was known about this mystery plasmid. Since then, our sample has denatured or was discarded accordingly. This semester, we (Aland Ali, Anh Nguyen, Rhiannon Giesegh, Mike Agneskiy) plan on farming and growing this plasmid to discover more of its characteristics. However, this week mainly consisted of a “bootcamp” of sorts that provided a refresher course of lab protocols, safety, storage, hygiene, and overall etiquette. Due to this, not much time was spent on beginning our projects until the end of the week. This time was spent creating TGY Agar plates and inoculating Deinococcus Aquaticus from freeze bag to farm our future plasmid. 


Procedure


To begin, 8 TGY Agar plates were created, labeled, and left overnight to ensure proper gel-like consistency. Next, Deinococcus Aquaticus from a freeze bag was acquired and set in an ice bucket to maintain more ideal temperature. This ice bucket, along with sterilized disposable inoculating loops were brought into a sterile environment containing a biosafety cabinet.  Surfaces of tables, plates and materials were sprayed with ethanol and wiped with standard napkins. The blower of the biosafety cabinet was turned on, the covering was lifted, and items were brought into the cabinet. It is imperative that once hands are in the cabinet, they do not come out (as it increases risk for contamination), Therefore, a second team member with sterile gloves assisted in the transfer of materials to the biosafety cabinet. Once all items were in the cabinet, inoculation of Deinococcus Aquaticus was continued as normal using a variation of spreads onto the 4-5 labeled TGY agar plates. This consisted of opening a disposable loop package from opposite end of loop, obtaining some of the Deinococcus Aquaticus sample once warm enough, and swiftly spreading the sample onto our TGY agar plates. New loops were used for each plate. Once complete, surfaces were cleaned, materials were discarded, and the UV light for the cabinet was turned on for >15 minutes to ensure sterilization and plates were stored in an incubator.  


Results 

Results of growth of Deinococcus Aquaticus are pending but should come by next week. Unused agar plates were placed in a refrigerator.





Discussion

As we wait for our samples to incubate, LB media was made for future inoculation of these potential colonies. Colonies are expected to be orange in appearance if all goes well. These colonies will be transferred aseptically to tubes of LB media and grown accordingly. The biosafety cabinet will not be used for these procedures as they are not our primary source of Deinococcus Aquaticus. 


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