Heh, when enough cells lyse, they make the solution isotonic to the rest! :) I imagine excessive lysis ups the salt contents and likelihood of electrode arcing though? Never did much EP myself.
Charles Vander Broek <charles.vanderbroek@gmail.com> wrote:
The only worry would be exposing the cells to such a hypotonic environment at room temperature would cause quite a lot of cell lysis. That being said, with an easy to use strain and enough cells loosing a few doesn't matter, especially if you are getting such good efficiency! Thanks for the info, will give that a try with a few strains myself! I have been meaning to try the sucrose method with a few Gram +ve cells, so I may try the DI Water as well!
On Thursday, October 17, 2013 10:58:27 PM UTC+1, Nathan McCorkle wrote:I've simply rinsed the overnight e.coli culture a few times with
sterile distilled water then electroporated, and proved better
transformation efficiency than using CaCl and heat shock.
Just started with the eppendorf full of overnight culture,
centrifuged, decanted and resuspended in DI H2O... repeated the pellet
rinsing twice more, added normal amount of plasmid DNA, electroporated
with GenePulser in a 0.1cm cuvette at 1.8kV.
See here for more details:
https://groups.google.com/d/msg/diybio/JcKyNIQGLIM/ u4qIOllQRnAJ
On Thu, Oct 17, 2013 at 10:26 AM, Charles Vander Broek
<charles.v...@gmail.com> wrote:
> Hi All,
>
> I just discovered DIY biology today and I am really excited for it's
> implications! I'm a microbiologist and I tend to try to find the fastest
> and easiest (read lazy) ways to work in the lab. I figure I will start by
> sharing my favorite method for making electrocompetent gram -ve bacteria.
> This method has worked very well for me (sometimes better than commercial
> cells) using E. coli, Pseudomonas spp., and Burkholderia spp. This method
> takes about 10-15 min, requires NO CHILLING, NO OD READINGS, and NO
> EXPENSIVE CHEMICALS which always pissed me off.
>
> Reagents:
> O/N culture of bacteria (Can be stationary or exponential phase, doesn't
> matter. About 6 ml of stationary phase E. coli is enough for 2x 50 ul
> transformations).
> DI water
> Sucrose (Now this is where it gets interesting. I have always used
> laboratory grade D-sucrose, but I think table sugar should do just fine as
> it is around 99.8-99.9% sucrose and should have low enough ion
> concentrations that it should work. If someone tries this let me know how
> it works and I will give it a try as well!)
>
> Equipment:
> 1.5ml microfuge tubes
> Table top Microfuge
> Optional: A larger cetrifuge and tubes for bulking up the procedure for
> large quantities of transformations.
>
> Procedure:
> 1. Prepare 300mM sucrose in the DI Water
> 2. Spin down the bacteria. 6ul can be split into 4x 1.5 ml microfuge tubes
> or spun in a larger tube. The spin should be enough to pellet the bacteria
> for removal of the supernatant (ie 13,000 RPM for 2 min in a tabletop
> microfuge)
> 3. Remove the supernatant and re-suspend the bacteria in 1 ml of 300 mM
> sucrose. The bacteria split in the 4 tubes can be combined at this point
> (suspend all 4 pellets in the same 1ml of 200 mM sucrose).
> 4. Pellet the bacteria at 13,000 RPM for 2 min in a table to microfuge.
> 5. Remove the supernatant and re-suspend the pellet well in 100 ul of 300 mM
> sucrose.
> 6. The bacteria are now ready to be electroporated. I usually split the
> pellet in two 50 ul alliquots and add about 100-200 ng of plasmid DNA to the
> 50 ul of bacteria.
> 7. I transform using a 1 cm cuvette at 1.8 kV.
>
> If you are getting sparks try adding one extra wash in sucrose. Also I
> suspend my DNA in DI water so if you are suspending in a solution with ions
> this may affect your electroporations.
>
> Good luck and happy trails! If you need any more info let me know!
>
>
> The paper that this detailed in! These guys made my life so much easier :):
>
> J Microbiol Methods. 2006 Mar;64(3):391-7. Epub 2005 Jun 28.
>
> A 10-min method for preparation of highly electrocompetent Pseudomonas
> aeruginosa cells: application for DNA fragment transfer between chromosomes
> and plasmid transformation.
>
> Choi KH, Kumar A, Schweizer HP.
>
> Source
>
> Department of Microbiology, Immunology and Pathology, Colorado State
> University, 1682 Campus Delivery, Fort Collins, CO 80523, USA.
>
> Abstract
>
> A rapid microcentrifuge-based method is described for preparation of
> Pseudomonas aeruginosa electrocompetent cells with up to 10,000-fold
> increased transformation efficiencies over existing procedures. This
> increased efficiency now enables the use of transformation for all
> applications requiring DNA transfer. These include transfer of chromosomal
> mutations marked with antibiotic resistance genes between P. aeruginosa
> strains, which solves the riddle of not having an efficient and reliable
> transduction procedure for this bacterium. Not surprisingly, the method also
> allows for very efficient transformation with replicative plasmids, with
> transformation efficiencies ranging from 10(7) to >10(11) transformants per
> microgram of DNA. Lastly, with efficiencies of up to >10(3) transformants
> per microgram of DNA the method replaces in most instances conjugation for
> the transfer of non-replicative plasmids used in gene replacement,
> site-specific gene integration and transposon mutagenesis experiments.
>
> PMID: 15987659 [PubMed - indexed for MEDLINE]
>
>
>
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-Nathan
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Sent from my Android device with K-9 Mail. Please excuse my brevity.
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