Addgene: Protocol – How to do a Bacterial Transformation

Please note: Your browser does not support the features used on Addgene's website. You may not be able to create an account or request plasmids through this website until you upgrade your browser. Learn more Please note: Your browser does not fully support some of the features used on Addgene's website. If you run into any problems registering, depositing, or ordering please contact us at help@addgene.org. Learn more Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids, even those designed for use in mammalian cells, carry both a bacterial origin of replication and an antibiotic resistance gene for use as a selectable marker in bacteria. Scientists have made many genetic modifications to create bacterial strains that can be more easily transformed and that will help to maintain the plasmid without rearrangement of the plasmid DNA. Additionally, specific treatments have been discovered that increase the transformation efficiency of the bacteria and make them more susceptible to either chemical or electrical based transformation, generating what are commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing. For the highest transformation efficiency, we recommend that you follow the instructions that came with your competent cells. Take agar plates (containing the appropriate antibiotic) out of 4°C to warm up to room temperature or place in 37°C incubator. Mix 1 to 5μl of DNA (usually 10pg to 100ng) into 20-50μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 seconds (45sec is usually ideal, but this varies depending on the competent cells you are using). Plate some or all of the transformation onto a 10cm LB agar plate containing the appropriate antibiotic. If you are not concerned with transformation efficiency (such as when you have a tube of plasmid DNA and just need to transform bacteria so that you can grow up more of the plasmid) you can save a lot of time by shortening or skipping many steps and will still get enough colonies for your next step. Remember that each of these shortcuts will reduce the efficiency of the transformation, so when higher efficiency is needed follow the complete protocol. Shorten or skip the outgrowth (for Ampicillin resistance it is ok to completely skip the outgrowth, for the other antibiotics it is a good idea to outgrow for at least 20-30 minutes) I get very few if any transformants when transforming large plasmids (>,10kb) or BACs, what can I do? Chemically competent cells are fast and easy to use, but are less efficient at taking up larger plasmids. If you need to transform large plasmids, it is a good idea to use electro-competent cells. Instead of relying on the heat-shock to cause the cells to take up the DNA, an electro-magnetic field is applied to the cell/DNA mixture to induce membrane permeability. To do this you will need to have access to an electroporator and the appropriate cuvettes. Follow the manufacturer's instructions for each. Check that you are plating on an LB Agar plate containing the correct antibiotic. The resistance gene on your plasmid must match the antibiotic on the plate. You should also add a positive control (many companies include a positive control plasmid with their competent cells) to ensure that your transformation procedure is working. Although it may be counter-intuitive, you will often get higher transformation efficiencies with less DNA, especially when using highly competent cells. If you used 100-1000ng of total DNA in a ligation you will often get more colonies if you use 1μl of a 1:5 or 1:10 dilution rather than 1μl directly. Addgene is a nonprofit plasmid repository. We archive and distribute high quality plasmids from your colleagues. Source.


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Last Modified: January 27, 2014 @ 12:00 am