Generic PCR

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Ideas behind PCR optimizations: In order to optimize PCRs, we need to ask the following questions:

— Do the primers actually work? To answer this, we use OneTaq polymerase until we find conditions that amplify exactly one band of the expected PCR product size.

— Do we want to clone something? For this, we need to use Q5 polymerase, which has a lower error rate (but is more expensive!). We also only want to try this out once we know that the primers work — we should optimize for OneTaq before moving on to Q5.


Generic PCR Recipe

For a 10ul reaction:

Reagent Volume
OneTaq Reaction Buffer (5X) 2.0ul
dNTPs (Nucleotides; 40 mM total) 0.2ul
Forward Primer (10uM) 0.75ul
Reverse Primer (10uM) 0.75ul
DNA template (< 50ng/ul) 1.0ul
OneTaq Polymerase (5U/ul) 0.1ul
Molecular-grade Water (from refrigerator) 5.2ul


PCR Machine Protocol, OneTaq

  1. 94° for 30 seconds (Use 5 minutes in the case of colony PCR to lyse the cell)
  2. 94° for 30 seconds (Denaturing temperature)
  3. Annealing Temperature for 30 seconds (see section below)
  4. 68° (Extension temperature; Use 60 seconds per 1kb of expected product)
  5. Go to step 2, and repeat 34 times (so, 35 cycles total)
  6. 68° for 5 minutes
  7. 12° hold


PCR Machine Protocol, Q5

  1. 98° for 30 seconds
  2. 98° for 10 seconds (Denaturing temperature)
  3. Annealing Temperature for 30 seconds (50° - 72°; see section below)
  4. 72° (Extension temperature; use 30 seconds per 1kb of expected product)
  5. Go to step 2, and repeat 34 times (so, 35 cycles total)
  6. 72° for 3 minutes
  7. 12° hold


Annealing Temperature

Use this link to get the melting temperature of a primer.

The annealing temperature for PCR is then this melting temperature that is systemically modified.

  • OneTaq annealing temperature should be 3 degrees below melting temperature of primers
  • Q5 annealing temperature should be 2 degrees above melting temperature of primers.


If you are working with new primers, use this protocol to find the ideal annealing temperature.

  • Initial test: Try a single 10ul reaction using OneTaq and an annealing temperature of 3 degrees below melting temperature of primers.
  • If you do not see a single, correctly-sized band, switch to a gradient PCR using 8 tubes across 10 degrees.
    • If there are more than one band in the initial test, use the original annealing temperature as the lowest temperature in the gradient
    • If there are no band in the initial test, use the original annealing temperature as the highest temperature in the gradient

Once we know that we can make a clean PCR reaction with OneTaq, switch to Q5 by adding 5 degrees of the annealing temperature.


Generic PCR Recipe with DMSO

Problematic PCR's? Try using 3%-10% DMSO. This will then change the annealing temperature, necessitating a gradient PCR across 15 degrees.

10% DMSO (1ul in 10ul reaction)