PCR
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.
Technical notes for standard PCR
In this post, you’ll learn troubleshooting tips and equipment information for PCR experiments.
Sample Preparation
To prepare samples for PCR, DNA containing the gene of interest is extracted from cells. DNA extraction is typically done through pre-made kits provided by companies like Qiagen. 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:
34uL Water
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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Thermal Cycling
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.
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- Perkin Elmer Chemagic 360
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| Stage | Time | Temperature |
|---|---|---|
| Melting | 3 min | 98 |
| 25 Cycles | 30 second | 98 |
| 25 Cycles | 30 sec (Annealing) | Depends on Tm |
| 25 Cycles | (15-30s/ kb being amplified) | 72 |
| Extension | 10 min | 72 |
| Holding | Infinite | 4 |
Collection of DNA
Collecting DNA at this stage can be done in one of two ways. The first method is via PCR cleanup. If the amplified fragment does not need to be 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. The solubilization buffer and centrifugation columns are provided in gel extraction kits available from companies like Qiagen.
Featured Products
- Perkin Elmer Chemagic 360
- Macherey-Nagel NucleoMag Pathogen Kits