Polymerase chain reaction (PCR) is a tool that exponentially reproduces target DNA sequences from free nucleotides in a controlled reaction.It is commonly used to amplify specific DNA sequences for use in cloning or detection and has become a staple in any laboratory using molecular biology techniques.
In this post, you’ll learn troubleshooting tips and equipment information for PCR experiments.
To prepare samples for PCR, DNA containing the gene of interest is extracted from cells. DNA extraction is typically done through pre-made kits. These kits contain buffers and columns that can be used to lyse the cells and extract DNA through centrifugation.
Another key component of sample preparation is the acquisition of DNA oligos that flank the DNA sequence of interest. More information on designing these DNA oligos can be found on the Cloning page. The designed oligos can be created synthetically in vitro or ordered from companies like IDT.These oligos will serve as the forward and reverse primers for the polymerase to latch onto and begin synthesizing double stranded DNA fragments.
A DNA polymerase will also be needed to catalyze the creation of double-stranded DNA. In the reactionDNA polymerases are critical components in PCR since they synthesize the new complementary strands from the single-stranded DNA templates. All DNA polymerases possess 5′→ 3′ polymerase activity. DNA polymerases must be included with free nucleotides to properly synthesize DNA.
An example of the composition of PCR-ready sample preparation:
10uL 5x Buffer specific to polymerase
1uL dNTPs(free nucleotides)
1.25uL Forward Primer
1.25uL Reverse Primer
2uL 1:1000 Template DNA containing gene of interest
0.5uL DNA polymerase such as Taq or Phusion
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Amplification is achieved by a series of three steps: (1) denaturation, in which double-stranded DNA templates are heated to separate the strands; (2) annealing, in which the primers bind to flanking regions of the target DNA; and (3) extension, in which DNA polymerase extends the 3′ end of each primer along the template strands. These steps are repeated, or cycled, 25–35 times to exponentially produce exact copies of the target DNA.
Note that melting and extension temperatures do not typically change from each experiment, but care must be taken to adjust the annealing temperature for each construct based on their primers. Each primer has an associative melting temperature that can be inferred from its DNA sequence. Most plasmid editors have this feature built in, otherwise there are online resources to provide the melting temperature based on the sequence given. These melting temperatures should be similar (within 5℃ of each other) and the sequences can be adjusted when designing to adjust the melting temperatures. The higher temperature should be preferred in each context.
A Sample of Cycling Conditions
|25 Cycles||30 second||98|
|25 Cycles||30 sec (Annealing)||Depends on Tm|
|25 Cycles||(15-30s/ kb being amplified)||72|
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Separated from other fragments of DNA that may be present in the sample, PCR cleanup can be done directly on the results from the PCR. PCR cleanup will isolate all DNA fragments within the sample. PCR cleanup kits are available from companies like Qiagen and function similarly to DNA extraction kits.
The other, more specific way of collecting the DNA fragment is through gel extraction. Using the gel that the PCR was confirmed on, the band that contains the DNA fragment of interest is visualized by UV light and physically cut from the gel. This gel piece is dissolved in solubilization buffer and the DNA is isolated via centrifugation into a separate tube for later use.
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1. Developing a primary culture
Primary culture refers to the cells that are isolated directly from the tissue of interest and proliferated until they reach confluence, or occupy all the available substrate. One method of acquiring cells for primary culture entails sampling from the tissue directly. Cells taken in this manner must be disaggregated using enzymatic or mechanical means before they are placed on the substrate.
After the primary culture reaches confluence, the cells have to be subcultured by transferring them to fresh growth medium. At this stage the cell culture is no longer considered primary and becomes secondary culture, or cell line. Cell lines derived from primary cultures are finite, which means they have a limited number of cell divisions that is a genetically determined. The loss of ability to proliferate is called senescence.
Another way of acquiring cell culture is by using an established cell strain. These are continuous, or immortalized, cell lines that have been mutated and lost the ability to undergo senescence. These cells can continuously divide and are optimal cell lines for prolonged studies. The most common immortalized cell lines used for primary culture include HeLa cells and HEK 293 cells, among others
2. Developing Cell Lines
Regardless of the cell line type, it is necessary to propagate the cell line by continuously passaging the cells into fresh media, a process known as splitting the cells. To split cells, cell media is aspirated via a vacuum and the cells are washed in warmed Phosphate Buffered Saline (PBS) to remove residual media. If the cells are anchorage-dependent and rely on adherence to a surface, the cells can be treated with trypsin, a chemical that causes cells to temporarily lose their adherence. Trypsin, however, is toxic and should not be applied for long.
Trypsin toxicity is neutralized by adding growth media. For mammalian cells, this growth media is typically DMEM supplemented with Fetal Bovine Serum (FBS) and antibiotics. From this solution, the media and cells can be partitioned, or split, into new cell culture plates. A common practice is to always maintain at least one plate for further splitting and use the rest to split into additional plates that will be used for experiments.
While the above example is for anchorage-dependent mammalian cells, a similar principle applies to other types of cells including those in suspension and spore colony culture. In suspension culture, cells from a developed culture may be split by transfer into fresh growth media. In colony culture, spores or colonies may be taken from a developed plate and streaked across fresh media plates
Different cells require different growth conditions. While conditions may vary from cell type to cell type, most cells are grown in incubators to maintain optimum growth conditions. Incubators have the ability to maintain an optimum temperature (37oC for most cells) and precise O2 and CO2 levels. Additionally, cells will require a growth medium that is unique to each cell type. Media contains the nutrients, hormones, and buffers for cells to grow and should be continually changed. The right media and incubation conditions are critical for cell growth.
3. Maintaining Cell Lines
Successful cell culture depends on keeping the cells free from contamination by microorganisms such as bacterial, fungi, and viruses. Nonsterile supplies, media, reagents, airborne particles, unclean incubators, and dirty work surfaces are all sources of biological contamination.
Aseptic technique is the best barrier to prevent contamination by invasive microorganisms and should always be maintained. Aseptic technique includes:
- Only opening cell culture in a sterile environment such as a cell culture hood with working airflow.
- Sterilizing the work area before and after each handling of the cells with proper cleaning reagents. 70% ethanol is the most common choice.
- Using gloves, lab coats, and other PPE as needed to prevent contamination of the samples and maintain personal safety.
- All media and solutions should be opened and/or mixed within a cell culture hood.
- Using only sterile glassware and disposable pipettes. Dispose of pipettes after each use.
- Opening media and other containers only when ready to use.
However, even with proper aseptic technique, contamination can happen. In these cases, the incubator and cell culture hood should be thoroughly cleaned to prevent other samples from being contaminated. If contaminated cells or media are critical to work, they may be treated with antibiotics to kill the contaminants - though care should be taken as antibiotics may cross-react with the cells of interest. In most cases, the contaminated cells and media should be thrown out and prepared anew.
Long-term storage of cells is a useful way to backup any experiments relying on a certain subculture or strain of cells. The most common method is Cryopreservation. Cryopreservation requires a surplus of cells be taken from cell culture and mixed with a protective agent, typically DMSO or glycerol, before being stored below –130°C.