From 2010.igem.org

Revision as of 15:38, 10 September 2010

Comments on the Protocols

Taq 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.

1. First, the two strands of the template DNA are separated (denatured) at 95°C, which is a high enough temperature to break the hydrogen bonds between the base pairs.
2. Next comes the annealing of the primers. The annealing 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.
3. Then, at 72°C, the Taq polymerase extends the annealed primer to create a copy of the DNA template. The process repeats.

Primers are short pieces of DNA that are complementary to the base strand. Since polymerase can only attach an incoming 5' nucleotide to an existing 3' end, a starting point for the polymerase to "attach to" has to exist. This is the end of the primer. As a result, the portion bound between the two primers is amplified.

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).

Transformable Competent Cell Induction

Bacterial Transformation

Restriction Digest

Ligation

Miniprep Plasmid Preparation (using GenElute)

Agarose Gel Electrophoresis

Making of Agarose Gels

Rehydration of Registry DNA

Construction Technique

Overnight Cultures

Glycerol Stock Preparation

LB Agar Plate Preparation

QIAprep Spin Miniprep Kit

Gel Extraction

Tips

• Shorten melting time and alternate between 30 seconds in heat plate and 30 seconds in vortex and look at solution in light to ensure gel is melted.
• Use clear 1.5 tubes so that its possible to observe the colour change caused Binding Buffer(XP2) when the pH is too high or too low
• Dry step after the addition of Binding Buffer(XP2) could help separate the SPW buffer and Binding Buffer(XP2) stages.
• Shorten the length of spin and increase the rpm for the addition of binding buffer and SPW steps and increase to 2 minutes for drying stages
• Ensure that the spin column is dry before transferring into the clean 1.5 mL tube

PCR Purification