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Team:Calgary/Notebook/Safety And Protocols/Comments
From 2010.igem.org
Comments on the Protocols
Safety and Protocols Main PageTaq Polymerase Chain Reaction
Theory
PCR amplifies a portion of DNA, as bounded by specific primers. The process mimics the replication of DNA in a regular cell.
- First, the two strands of the template DNA are separated at 95°C, which is a high enough temperature to break the hydrogen bonds between the base pairs.
- Next comes the annealing of the primers. This temperature is extremely specific to the primer being used. There are many programs out there that can help you determine the annealing temperature of the primer.
- Then, at 72°C, the Taq polymerase extends the annealed primer to create a copy of the DNA template. The process repeats.
Taq polymerase is harvested from bacteria that live in hot springs, and it is used because it is functional at relatively high temperatures. Its optimal functioning temperature is at 72°C. Otherwise, the denaturation temperatures (at 95°C) will destroy the polymerase and cause the PCR to fail. MgCl2 is a salt that allows Taq polymerase to function.
Because polymerase can only go so fast, you should allow approximately 1 minute for every 1000 base pairs to be amplified, to ensure that the strand is fully copied before the cycle repeats. Step 5 is the final extension time, which ensures that all strands are extended completely. Then the PCR product can be held as long as you need it to be held at 4°C.
Tips
- For the Master Mix, you should add water in two portions (half added before all other reagents and half added after all other reagents).
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