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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