APPENDIX 3D Direct PCR of Intact Bacteria (Colony PCR) Michael E. Woodman1 1Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky ABSTRACT This protocol describes an efficient method for screening intact bacteria for the presence of desired DNA sequences using polymerase chain reaction (PCR). This method is commonly referred to as colony PCR. Curr. Protoc. Microbiol. 9:A.3D.1- A.3D.6. C 2008 by John Wiley & Sons, Inc. Keywords: colony PCR bacteria screen BASIC PROTOCOL PCR AMPLIFICATION OF DNA FROM A BACTERIAL COLONY (COLONY PCR) This unit describes a method for screening individual bacterial colonies for specific DNA sequences using the polymerase chain reaction (PCR see Kramer and Coen, 2001). This procedure, commonly referred to as colony PCR, is advantageous because it is a very quick and easy way to screen a large number of bacteria to determine which bacteria contain a particular DNA sequence, without first having to purify DNA from all of them. This is especially useful when screening colonies after transformation with recombinant plasmids or after targeted mutagenesis. Colony PCR can be effectively used not only to identify clones with an insertion or deletion, but also to determine the orientation of a DNA insertion, which may be important for proper transcription and translation of the insert. Furthermore, the method can be used to amplify a desired DNA fragment for subsequent sequencing or cloning. While colony PCR is most commonly used in the laboratory to screen transformed E. coli, DNA sequences from virtually any bacterial species can be detected and/or isolated regardless of location on the chromosome or a plasmid. Materials 10�� PCR buffer: supplied with DNA polymerase or see APPENDIX 2A 2.5 mM (each) dNTP mix: supplied with DNA polymerase or see APPENDIX 2A 30 ��M oligonucleotide primer 1 stock (see Critical Parameters) 30 ��M oligonucleotide primer 2 stock (see Critical Parameters) 5 U/��l Taq DNA polymerase, heat-stable (e.g., Takara Taq, Fisher) Distilled H2O, sterile MgCl2 (if required) Gel electrophoresis loading buffer (see recipe) 200-��l thin-walled PCR tubes (or other appropriate for thermal cycler) Toothpicks, sterile Thermal cycler Additional reagents and equipment for performing agarose (Voytas, 2000) or polyacrylamide (Chory and Pollard, 1999) gel electrophoresis Procedure 1. Prepare the master mix for PCR amplification (e.g., 100 ��l for ten tubes of 10 ��l each): Current Protocols in Microbiology A.3D.1-A.3D.6, May 2008 Published online May 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/9780471729259.mca03ds9 Copyright C 2008 John Wiley & Sons, Inc. Commonly Used Techniques A.3D.1 Supplement 9

Direct PCR of Intact Bacteria (Colony PCR) A.3D.2 Supplement 9 Current Protocols in Microbiology 10 ��l of 10�� PCR buffer containing 15 mM MgCl2 8 ��l of 2.5 mM (each) dNTP mix 5 ��l of 30 ��M primer 1 5 ��l of 30 ��M primer 2 1 ��l of 5 U/��l heat stable Taq DNA polymerase 71 ��l sterile distilled water. The volume of master mix prepared will depend upon the number of reactions and volume of each reaction. For example, if one is performing 20 reactions of 10 ��l each, 200 ��l master mix will be required. It is best to make a little extra master mix, since pipettors are often slightly inaccurate. The MgCl2 is often already included in the PCR buffer supplied with the DNA polymerase. If not, the appropriate amount must be added. Optimum MgCl2 concentrations for each PCR reaction may vary and can range from 1.5 mM to 4.5 mM (see Kramer and Coen, 2001). A high-fidelity Taq polymerase (e.g., Takara Ex Taq, Fisher) should be used if the PCR product is intended for use beyond simple screening, e.g., for DNA sequencing or later cloning into a vector for recombinant protein purification. 2. Dispense the master mix into the PCR tubes. 3. Using a sterile toothpick for each colony, remove a small amount of a bacterial colony directly from the plate to be tested and place in a PCR tube with master mix. Be certain to touch just one colony and avoid colonies very close to each other, otherwise this could contaminate the results. The amount of bacteria should be just barely visible to the unaided eye. PCR amplification will not be efficient if too few bacteria are transferred into the PCR reaction, while too many bacteria may cause amplification inhibition from other bacterial components. Experience will allow the researcher to know the proper amount of bacteria to allow for efficient PCR amplification. Since some reactions may fail, it is a good idea to simultaneously screen 10 to 20 colonies. Colonies may be needed for further study, so spotting each tested colony onto a plate divided into a numbered grid (Riley et al., 2008 also see APPENDIX 4A) is also recom- mended. 4. Carry out PCR using conditions appropriate for the DNA being tested, with the following amplification cycles as a guideline: Initial step: 5 min 94���C (bacterial lysis/ denaturation) 25 cycles: 30 sec 94���C (denaturation) 30 sec 50���C-55���C (annealing) 1 min 68���C-72���C (extension) Final extension: 5 min 72���C. A thermal cycler with a heated lid is recommended for convenience. Otherwise, overlaying the reaction mixture with mineral oil will be required (see thermal cycler instruction manual and Kramer and Coen, 2001). Twenty five cycles should be sufficient for production of a detectable amplicon. It is important to note that the ideal annealing temperature will vary depending on the GC content of the primers being used. Greater primer GC content requires higher annealing temperatures for specificity. Additionally, a general rule of thumb for PCR is to allow 1 min of extension time for every 1 kb of amplified DNA product. For a detailed description of the stages included in the PCR reaction see Kramer and Coen (2001).