Yeast colony PCR is a variation of the colony PCR technique that is used to amplify DNA from a single yeast colony. Like regular colony PCR, yeast colony PCR involves transferring a single colony of yeast to a PCR tube, adding PCR buffer, primers, and other reagents, and then running the reaction in a thermal cycler to amplify the target DNA. The amplified DNA can then be visualized on an agarose gel or stored for future use.
Yeast colony PCR is useful for researchers who want to study the genetic makeup of a particular yeast strain, or for clinicians who want to identify the species of yeast causing an infection.
What is PCR?
PCR, or polymerase chain reaction, is a technique used in molecular biology to make many copies of a specific DNA sequence. This technique is widely used in research, forensic analysis, and medical diagnostics.
To understand how PCR works, it’s helpful to know a little bit about DNA. DNA, or deoxyribonucleic acid, is the genetic material that makes up all living organisms. It’s made up of long chains of units called nucleotides, which are arranged in a specific sequence to carry genetic information.
In PCR, a small piece of DNA is first isolated and placed in a solution with special enzymes and other reagents. The solution is then heated to separate the two strands of the DNA molecule into single strands. Primers, which are short pieces of RNA, bind to specific sequences on the single strand DNA.
Once the primers are in place, the solution is cooled and the enzymes go to work. They use the primers as a starting point to make many copies of the target DNA sequence. This process is repeated multiple times, resulting in a large number of copies of the original DNA sequence.
In summary, PCR is a powerful technique that allows researchers to make copies of a specific DNA sequence, enabling them to study and analyze that sequence in detail. This technique has many important applications in molecular biology and other fields.
How is colony PCR different from regular PCR?
Colony PCR is a variation of the PCR technique. The main difference between colony PCR and regular PCR is the way in which the DNA sample is prepared.
In regular PCR, the DNA sample is extracted from a biological sample and then purified to remove any contaminants. The purified DNA is then used as a template for the PCR reaction.
In contrast, colony PCR uses cell colonies as the source of the DNA template. A colony is a group of organisms that has grown on a solid media. The cells in the colony are picked up using a inoculating loop, put into PCR reagents, and used as the template for the PCR reaction.
Overall, the main difference between colony PCR and regular PCR is the way in which the DNA sample is prepared, with colony PCR using cell colonies as the source of the DNA template.
Typical procedures for yeast colony PCR
To perform a yeast colony PCR, you first need to prepare your samples. This involves taking a small sample of yeast cells from a colony and suspending them in a buffer solution. Next, you will need to set up the PCR reaction by combining the sample with DNA polymerase (Taq), primers that flank the target sequence, and nucleotides. The PCR reaction is then amplified using a thermal cycler, which heats or cools the reaction mixture to allow the DNA polymerase to make copies of the target sequence.
The Taq polymerase enzyme is named after the thermophilic bacteria Thermus aquaticus. This enzyme is able to function at high temperatures, which makes it ideal for use in PCR reactions.Wikipedia
After the PCR reaction is complete, the resulting amplified DNA can be analyzed using a variety of techniques, such as gel electrophoresis or DNA sequencing.
A protocol for yeast colony PCR.
Joe Horecka and Angela M. Chu (2017) described a protocol that allows for efficient and successful amplification of DNA from yeast colonies. The authors tested this method using several different yeast strains and found that it consistently produced high-quality PCR products. They also compared it to traditional methods of PCR amplification and found that it was significantly faster and more efficient.
Preparing heat-popped yeast cells.
- Aliquot 50 µL of water into each 0.2 mL PCR tube.
- Pick up a small amount of yeast cells and transfer it to the water.
- Avoid touching the agar. Swirl the tube to release the cells from the pipet tip. Cap the tubes and gently vortex to suspend the cells.
- Place the tubes in a PCR machine and run a program of 99°C for 5 minutes, followed by a hold at 4°C.
- When done, remove the tubes from the machine and quick-spin them. Place the tubes on ice until they are ready to use, or freeze them at -20°C for later use.
PCR using heat-popped cells.
To continue with PCR, you can simply follow the same steps as with any template (plasmid or genomic DNA). In my experiment, I run 50 µL PCRs with Platinum™ II Hot-Start Green PCR Master Mix (Thermo Scientific):
- 25 µL Platinum II Hot-Start PCR Master Mix (2X)
- 1.25 uL each primer (0.5 µM each)
- 20.5 µL nuclease-free water
- 2 µL heat-popped yeast
The typical order for filling PCR tubes is to first add the master mix, then the primers (if not already included in the master mix), and finally, 2 µL of vortexed, heat-popped yeast. Quick-spin the samples using a mini centrifuge (for PCR tubes) or a centrifuge with a plate rotor (for PCR plates).
Run PCR with the following cycle:
94C, 30 sec denature 94C, 15 sec | 60C, 15 sec | 35 cycles PCR 68C, 30 sec | 68C, 1 min final extension 4C, hold end
Remove tubes from PCR machine and quick-spin.
In conclusion, colony PCR is a convenient and efficient method for performing PCR on yeast colonies. By eliminating the need for time-consuming cell lysis and DNA extraction steps, colony PCR provides researchers in the field of yeast genetics with a valuable tool for amplifying and analyzing genes of interest.
- Joe Horecka and Angela M. Chu 2017. Yeast Colony PCR: It doesn’t get any easier than this!. protocols.io