PCR technology is similar to the natural replication process of DNA, and its specificity relies on oligonucleotide primers complementary to both ends of the target sequence. PCR consists of three basic reaction steps: denaturation, annealing, and extension. This reaction process is carried out in the PCR reaction container. With the continuous development of the gene amplification instrument, the reaction container has also experienced the gradual development of the centrifuge tube from 1.5ml to 0.2ml (0.25ml) to the thin-walled 0.2ml dedicated for PCR. , 0.1mlpcr tube. With the increase in the number of PCR amplification reactions, high-throughput gene amplification containers for PCR strips and PCR plates are gradually formed.
PCR consists of three basic reaction steps: denaturation-annealing-extension
Denaturation of template DNA:
After the template DNA is heated to about 94°C for a certain period of time, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated, so that it can be combined with the primer to prepare for the next round of reaction.
Annealing (renaturation) of template DNA with primers:
After the template DNA is denatured into a single strand by heating, and the temperature is lowered to about 55°C, the primers are paired with the complementary sequence of the single strand of the template DNA.
Extension of primers:
Under the action of Taq, the DNA template-primer conjugate uses dNTP as the reaction raw material to synthesize a new semi-reserved replication chain complementary to the template DNA chain according to the principle of base pairing and semi-reserved replication.
Basic Principles of PCR Technology
The technology relies on an enzymatic synthesis reaction of DNA polymerase in the presence of template DNA, primers and four deoxyribonucleotides.
DNA polymerase uses single-stranded DNA as a template to initiate synthesis with a small piece of double-stranded DNA. One or two synthetic oligonucleotide primers are combined with a complementary sequence in the single-stranded DNA template to form a partial double-stranded DNA.
Under a suitable temperature and environment, DNA polymerase adds deoxymononucleotide to the 3´-OH end of the primer, and uses this as the starting point to extend along the 5´→3´ direction of the template to synthesize a new DNA complementary strand .
The difference between PCR tubes and centrifuge tubes
PCR tubes:
The PCR reaction plate is 96-well or 384-well, which is specially designed for batch reactions. The principle is that the throughput of the PCR machine and the sequencer is generally 96 or 384.
Centrifuge tube:
The centrifuge tube is not necessarily a PCR tube. There are many kinds of centrifuge tubes according to their capacity. The commonly used ones are 1.5ml, 2ml, 5ml, 15 or 50ml, and the smallest one (250ul) can be used as a PCR tube.
How to choose PCR tubes
We chose to place the PCR tubes in the hope of choosing the most accurate location for the temperature.
The most accurate position of the temperature should be the position above the temperature sensor under the module (regardless of the inaccurate temperature of the sensor).
The position of the temperature sensor of different PCR instruments is different.
For example, AB's 2720 has only one in the middle, MJ's 200 has three sensors, which are lower left, middle and upper right, Eppendorf's should be in row A or H, and Dongshenglong 811 has three temperature sensors, B1-2 should be selected , C1-2, C6-7, D6-7, B11-12, C11-12, because these points are temperature punctual points.
