Colony [[Polymerase chain reaction (Taq)|PCR]] is a variation of PCR that supplies a small amount of a microbial colony as the template of a PCR reaction. This in contrast to "standard" PCR methods that supply isolated DNA as the template, such as purified plasmid or the product of a previous PCR. This method has two common applications: 1. To evaluate colonies for the size, presence, or absence of a particular amplicon 2. To amplify a desired gene or region of the microbe's genome for downstream use While this method is often more rapid than more time-intensive methods such as gDNA extraction, it is also oftentimes less reliable. Several factors influence the reliability of this method: 1. The age of your colonies; fresh cells are better 2. The number of copies of your target DNA in each cell; multi-copy plasmids perform best, genomic targets perform worst 3. The amount of material you add to the reaction; too much cellular debris will inhibit the reaction Because of these factors, **it can be advantageous to have a high-quality gDNA sample of your background strain on hand to act as a positive control** for amplification, allowing you to distinguish between failure of the PCR due to preparation issues versus colony DNA quality. Choose one of the two following methods to perform a colony PCR. If neither procedure is working, I recommend considering [[Gram positive gDNA extraction|this protocol]]. ## Option 1: Lysis This is the more robust option, owing to the removal of most cellular debris. 1. [ ] Label a strip of PCR tubes according to the colonies you wish to test, filling each with 100 µL of PCR water. 2. [ ] In the biosafety cabinet, use a sterile loop to inoculate each tube with the corresponding colony; an entire colony's worth of material is appropriate.[^1] Use a pipette to disturb the colony, after adding it. 3. [ ] Use a thermocycler to heat the tube to 95°C for 5 minutes. 4. [ ] Transfer the contents of each tube to a microcentrifuge tube. 5. [ ] Spin the microcentrifuge tubes at 12,000 rcf for 5 minutes. 6. [ ] Transfer the supernatant into a fresh set of PCR tubes, clearly labeled, taking care to not disturb the pellet. Keep this DNA lysate on ice as your prepare your PCR reaction, or freeze it if you are not performing the reaction immediately.[^2] 7. [ ] When you prepare your PCR reaction, transfer 1 µL of this lysate as your template per 50 µL PCR reaction. ## Option 2: Direct addition This is a less reliable method that is predicated on the use of fresh cells in the midst of their growth phase, removed from the incubator during or within a just few hours prior of this procedure. Additionally, primer quality (e.g. binding affinity and annealing temperature) is more important for this method as they must withstand additional interference from cellular debris. Even if both of these conditions are met, this method can still fail — use it at your own risk. 1. [ ] After preparing the individual PCR reaction tubes, inoculate each PCR tube with a fraction of a single colony; a barely visible amount equivalent to the tip of a toothpick. 2. [ ] Start the PCR immediately, with an initial denaturation period increased to between 1- and 10-minutes.[^3] [^1]: If you are passaging this colony at the same time, passage the colony onto the new media first, using the same loop, then add the bulk of the material to the PCR tube. [^2]: DNA in this solution should not be considered stable, as this protocol does not take any steps for removal or inactivation of DNase enzymes that could degrade the DNA. Therefore, this lysate should not be stored for long periods without further cleanup (in which case a proper gDNA extraction protocol is likely appropriate). [^3]: It may be desirable to test multiple denaturation times, depending on your species. Once you have a working initial denaturation period, use that time consistently.