Harvesting E.coli from LB Agar Plates

E.coli will be harvested from LB Agar plates. The cells will be centrifuged at 8000 rpm for 10 min to pellet. The supernatant will be removed and the cells resuspended in 5 mL TE Buffer. In the follow up video DNA will be Extracted and purified.

[@firasbn1582]

Thank you

[@ayaqz3144]

thank you from iraq

[@dinornis]

Is this method suitable for fungal colonies? (I guess the thing that makes me anxious is sending spores everywhere, but under a biosafety cabinet?)

[@ProfessorDrewCollop]

Thanks for the question Elle. I personally have no experience harvesting fungi, but a colleague of mine down the hall from my office is an expert. If you email me your questions to [e-mail] I will forward your questions on to him.

[@torshaghosh3853]

That scrapper has any specific name? Also does this process interfere with biofilm or viability of the strain? I want to harvest live bacteria with intact biofilm.

[@ProfessorDrewCollop]

Thanks for your question Torsha. I believe the technical name for this is Stainless Steel Cell Spreader. There are disposable plastic ones call Delta Cell Spreaders. Often in the lab we create fresh ones by melting a glass rod or pasteur pipette into a hockey stick shape. That is why I was referring to the cell spreader as a hockey stick (and the fact that I am Canadian). We ethanol flame them to sterilize them, then used it to harvest bacteria. I have never had an issue with the cell spreader damaging the viability of the strain. Unfortunately, I do not have an answer for you, in terms of biofilm. I do not have experience harvesting intact biofilm. Perhaps if you reached out to the manufacturer they could provide you with this information. Best of luck.

[@marie-joework9522]

Can this also be used to extract proteins or only DNA?

[@ProfessorDrewCollop]

Thanks for the question. This protocol is for harvesting of the E.coli from the plate. I don't see why you could not use this to harvest E.coli for your protein extraction protocol. I am using TE buffer to try to limit DNase activity, so that might not be necessary. Please note that the DNA extraction protocol I use in the next video is specific for DNA. There is a different protocol for protein extraction.

[@syed-wd8ib]

what is the purpose of using TE buffer for resuspention

[@ProfessorDrewCollop]

The "T" stands for Tris. It is the buffering agent to maintain the pH of the solution. The "E" stands for ETDA. It is a chelator (grabs them out of the solution and holds onto them) of cations such as Mg+ and Ca+. These cations are required for enzymes, such as DNase (an enzyme to breaks down DNA), to be active. As we are attempting to isolate DNA, we would like to limit the effects of DNase.

[@anneshb6397]

Sir how long we kept it for centrifugation

[@ProfessorDrewCollop]

Hi Annesh. I am sorry but I do not understand your question. Are you asking for the speed and duration of the spin?