Team:Calgary/Notebook/Safety And Protocols/Comments

From 2010.igem.org

Revision as of 18:15, 10 September 2010 by Jdchoo (Talk | contribs)

Comments on the Protocols

Safety and Protocols Main Page

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).

Close Window

Transformable Competent Cell Induction

Close Window

Bacterial Transformation

Close Window

Restriction Digest

Close Window

Ligation

Close Window

Miniprep Plasmid Preparation (using GenElute)

Close Window

Agarose Gel Electrophoresis

Close Window

Making of Agarose Gels

Close Window

Rehydration of Registry DNA

Close Window

Construction Technique

Close Window

Overnight Cultures

Close Window

Glycerol Stock Preparation

Close Window

LB Agar Plate Preparation

Tips

  • Tape up plate sleeves if planning to reuse the sleeves
Close Window

QIAprep Spin Miniprep Kit

Tips

  • Drying for 2 minutes instead of 1 minute can ensure that all of the wash solution is removed
  • When doing the aliquot step, put the pipette tip directly on the filter and eject in the center of the filter
  • Wait 15 minutes after adding the water onto the filter in the aliquot step
Close Window

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
Close Window

PCR Purification

Close Window