PCR amplification of genomic or plasmid DNA

PCR from genomic DNA or a plasmid template
Below are two protocols, both are known to work.
My two cents (Caroline): Using Vent (condition A) works for most (>90%) parts. However, there have been few parts for which I couldn’t get pcr products using condition A. I have been able to pcr out these difficult parts using Pfx (condition B) — Pfx has worked for *all* pcr reactions I’ve tried.
Condition A: Vent polymerase
Materials Required

  • 10x ThermoPol buffer
  • Vent DNA polymerase
  • 25 mM dNTPs
  • template DNA
  • 100 µM forward primer
  • 100 µM reverse primer


  1. Design your primer per the PCR primer design general instructions.
  2. Resuspend each primer in Tris buffer pH 8.0 or distilled water to 100 µM.
  3. Mix, adding the enzyme last:
  • 5 µL 10x ThermoPol buffer
  • 0.4 µL 25 mM dNTPs
  • 0.5 µL 100 µM forward primer
  • 0.5 µL 100 µM reverse primer
  • ≤1 µL plasmid DNA or 2 µL genomic DNA
  • 1 µL Vent DNA polymerase
  • distilled water to 50 µL total volume
  1. Use the following thermocycling program:
  • Start: 95 °C for 2 min. (melt)
  • Cycle 95 °C for 0.5 min (melt)
  • Tm minus 5 °C for 0.5 min. (anneal)
  • 74 °C for (# bp/1000) min. (extension) – no less than 0.5 min.
  • No. of Cycles: 30
  • End: keep at 4 °C forever

Condition B: Pfx Polymerase

Note: the conditions described below do not result in 1x amplification buffer, etc. In my experience these non-standard conditions perform better than the standard ones.
Materials Required

  • Platinum Pfx polymerase (Invitrogen catalog #11708-013)
  • 10 mM dNTPs in water (this means [dATP]=[dCTP]=[dGTP]=[dTTP]=10 mM)
  • template DNA
  • 100 μM forward primer in water
  • 100 μM reverse primer in water


  1. Resuspend each primer in Tris buffer pH 8.0 or distilled water to 100 µM.
  2. Mix, adding the enzyme last:
  • 3 µL primer mix (10µM of each primer)Note:I typically make this primer mix by adding 1 µL of the 100 µM forward primer, 1 µL of the 100 uM forward primer, and 8 µL of sterile water.
  • 0.8 µL template DNA
  • 25 µL 10X PFx amplification buffer
  • 3 µL 10mM dNTPs (each dNTP is 10 mM)
  • 2 µL 50mM MgSO<sub>4</sub>
  • 30 µL 10X PFx enhancer buffer
  • 34.2 µL water (to 100 µL)
  • 2 µL PFx DNA polymerase
  1. PCR Program
  • Start: 94 °C for 5 min. (melt)
  • Cycle 94 °C for 15 sec (melt) (cycle start)
  • 55 °C for 0.5 min. (anneal)
  • 55 °C is a suggested annealing temperature. However, VectorNTI will suggest an annealing temperature (TaOpt) when it generates a primer set.

If you have a series of pcr reactions, use the lowest annealing temperature of the group.

  • 68 °C for 1 min/kb (no less than 0.5 min) (extension)
  • 68 °C for 7 min (cycle end)
  • No. of Cycles: 35
  • End: keep at 4 °C forever


== C. Optional: Restriction Digest ==
* If you are going to digest your PCR product for ligation into a plasmid, I recommend doing the digestion before doing gel analysis. Doing the digest before the gel analysis will not allow you to identify cut vs. uncut DNA (typically this is only a few bp difference), but it will allow you to exclude any spurious PCR products that contain those restriction sites.
* See the [[NanoBio: Restriction Digest| restriction digest page]] for a detailed procedure of digesting PCR products.

== D. Gel analysis and purification of PCR products ==
* Run the (digested) PCR reaction on an appropriate percentage agarose gel.
* Image the gel, and identify the bands of interest.
* Cut out the band of interest.
** Wear a lab coat, gloves, and UV-protective googles to avoid accidental UV exposure, i.e. a bad sunburn.
** If using the Chemi Doc, slide out the UV transilluminator completely & insert the plexiglass protective shield in the holder at the front of the tray.
** Using a disposable scalpel, cut as small a piece of gel as possible that contains the central DNA band region. Be conservative.
** Using tweezers, place the gel pieces into labeled tubes.
* Image the gel after you cut and remove the bands. This image documents that you cut out the correct band.
* Gel extract the DNA using Qiagen’s PCR purification kit. Use the instructions for gel purification, and elute with 30 µL.
** If you end up with a reasonable chunk of gel, measure its weight in advance of doing the purification, and follow the guidelines for how much buffer to dissolve the gel in. If you do not, agarose (we suspect) can end up precipitating in your ‘purified’ DNA.
* Measure the A<sub>260</sub>/A<sub>280</sub> ratio of your purified DNA. It should be ~1.5 . If it is ~1.0, re-purify the DNA by doing an additional PCR purification.

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