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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 such as BLAST primer and GCK.
3. Then, at 72°C, the Taq polymerase extends the annealed primer to create a copy of the DNA template. The process repeats. Note: you can use Pfu or other polymerases with exonuclease activity in order to ensure that there are no mutations are added.

Primers are short pieces of DNA (approximately 20-30 bases long) 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).
• Make sure you spin down the mixture with centrifuge or microfuge before you add Taq

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Preparation of Competent cells

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Bacterial Transformation

You may leave the cells shaking for longer than an hour but not overnight. Leaving the cells in the shaker for about 2-4 hours allows the cells to grow and recover well. This gives high transformation efficieny with lots of colonies. Close Window

Restriction Digest

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Ligation

Quick ligase is expensive, you can use quick ligase buffer with regular T4 ligase and ligate at room temperature for 15-20 minutes and it will do teh same trick! Close Window

Miniprep Plasmid Preparation (using GenElute)

Tips

• When you are specing your DNA, make sure you use the elution water/ buffer as your blank.
• When spinning your DNA after lysing and neutralization you can spin for 15-20 minutes. This allows the debris to stick to the side of the tube allowing transferring the supernatant with the plasmid much easier and contaminant free. Close Window

  Agarose Gel Electrophoresis

  Tips

  If you are looking for a big band higher up in the ladder, run the gel at a higher voltage for a shorter period of time. Ethidium bromide wash if necessary. You don't always have to prepare the 20 μL of solution, you can add appropriate amount of DNA usually 3 μL and then add 2 μL dye and just run teh entire mixture in the gel. Make sure you are careful and there are no bubbles in the well, or in your pipette tip. That would move the DNA out of the gel. Close Window

  Making of Agarose Gels

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  Tips

  • When looking for large bands, make gels that are higher than one percent and run at a higher voltage, do the opposite for smaller sized bands.

  Rehydration of Registry DNA

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  Construction Technique

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  Overnight Cultures

  Tips

  • Make a control tube with your respective antibiotic. Do not put a colony in there and see if there is any growth. This will ensure that your antibiotics are working.
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  Glycerol Stock Preparation

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  LB Agar Plate Preparation

  Tips

  • Tape up plate sleeves if planning to reuse the sleeves
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  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
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  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
  • You may use isopropanol to dilute the QG solution after the gel is melted. This increases the yield of DNA
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  PCR Purification

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