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A strip of eight PCR tubes, each containing a 100 μL reaction mixture Placing a strip of eight PCR tubes into a thermal cycler. The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study.
Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. [2]
Chip-based Digital PCR (dPCR) is also a method of dPCR in which the reaction mix (also when used in qPCR) is divided into ~10,000 to ~45,000 partitions on a chip, then amplified using an endpoint PCR thermocycling machine, and is read using a high-powered camera reader with fluorescence filter (HEX, FAM, Cy5, Cy5.5 and Texas Red) for all ...
Plasmid Cloning Guides Molecular Cloning Guides —References to help scientists design plasmid cloning experiments, including tutorials on restriction enzyme digestion and PCR-based cloning. Molecular Cloning Protocols —Specific protocols for a variety of plasmid cloning techniques, such as isolation of bacterial colonies, DNA purification ...
Mixing this destination plasmid with the entry clones and the LR clonase mix will allow for recombination to occur between the attR and attL sites. A destination clone is produced, with the fragment of interest successfully inserted. The lethal gene is inserted into the original vector, and bacteria transformed with this plasmid will die.
Polymerase cycling assembly (or PCA, also known as Assembly PCR) is a method for the assembly of large DNA oligonucleotides from shorter fragments. The process uses the same technology as PCR, but takes advantage of DNA hybridization and annealing as well as DNA polymerase to amplify a complete sequence of DNA in a precise order based on the single stranded oligonucleotides used in the process.
There are two fundamental differences between the methods. One is that molecular cloning involves replication of the DNA within a living cell, while PCR replicates DNA in the test tube, free of living cells. The other difference is that cloning involves cutting and pasting DNA sequences, while PCR amplifies by copying an existing sequence.
The first step in Gateway cloning is the preparation of a Gateway Entry clone. There are a few different ways to make entry clone. Gateway attB1 and attB2 sequences are added to the 5' and 3' end of a gene fragment, respectively, using gene-specific PCR primers and PCR amplification. The PCR amplification products are then mixed with a propriet
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