Home Protocols PCR Protocol for OneTaq® Quick-Load® DNA Polymerase (M0509)

PCR Protocol for OneTaq® Quick-Load® DNA Polymerase (M0509)

Overview

PCR 

The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification. Taq DNA Polymerase is an enzyme widely used in PCR. OneTaq Quick-Load DNA Polymerase allows for greater amplification sensitivity across a wide variety of amplicons. The following guidelines are provided to ensure successful PCR using New England Biolabs’ OneTaq Quick-Load DNA Polymerase. These guidelines cover most routine PCR. Specialized applications may require further optimization.


Protocol

Reaction setup: 

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94°C).

Add to a sterile thin-walled PCR tube:
Component 25 μl reaction 50 μl reaction Final Concentration

5X OneTaq Quick-Load Buffer or

5X OneTaq Standard Reaction Buffer 

5 µl

10 μl

1X

10 mM dNTPs (#N0447)

0.5 µl

1 μl

200 µM

10 µM Forward Primer

0.5 µl

1 μl

0.2 µM

10 µM Reverse Primer

0.5 µl

1 μl

0.2 µM

OneTaq Quick-Load DNA Polymerase

0.125 µl

0.25 µl

1.25 units/50 µl 
PCR*

Template DNA

variable

variable

< 1,000 ng

Nuclease-free water

to 25 µl

to 50 µl

*For amplicons between 3–6 kb, use 2.5–5 units/50 µl rxn

Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.


Transfer PCR tubes to a PCR machine and begin thermocycling:


Thermocycling conditions for a routine PCR: 

STEP  TEMP  TIME 

Initial Denaturation

94°C

30 seconds

30 Cycles

94°C
45-68°C
68°C

15-30 seconds
15-60 seconds
1 minute per kb

Final Extension

68°C

5 minutes

Hold

4-10°C

 

General Guidelines: 

  1. Template: 

Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:
    DNA Amount

    genomic

    1 ng–1 µg

    plasmid or viral

    1 pg–1 ng
  2. Primers:

    Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as PrimerSelect™ (DNAStar Inc., Madison, WI) and Primer3 can be used to design or analyze primers. The final concentration of each primer in a PCR reaction may be 0.05–1 µM, typically 0.2 µM.

  3. Mg++ and Additives:

    Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with OneTaq Quick-Load DNA Polymerase. The final Mg++concentration in 1X OneTaq Quick-Load Buffer or Standard Reaction Buffer is 1.8 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.2 mM increments using MgCl2 (NEB #B9021).

  4. Deoxynucleotides:

    The final concentration of dNTPs is typically 200 µM of each deoxynucleotide.

  5. OneTaq Quick-Load DNA Polymerase Concentration:

    We generally recommend using OneTaq Quick-Load DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 µl reaction) for amplicons up to 3 kb. The optimal concentration of OneTaq Quick-Load DNA Polymerase may range from 5–100 units/ml (0.25–5 units/50 µl reaction). For specialized applications, including 3–6 kb amplicons, 2.5–5 units/50 µl reaction is recommended. Note that in some cases increasing the amount of enzyme in the reaction can be inhibitory.

  6. Denaturation:

    An initial denaturation of 30 seconds at 94°C is sufficient to amplify most targets from pure DNA templates. For difficult templates such as GC-rich sequences, a longer denaturation of 2–4 minutes at 94°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 2–5 minute incubation at 94°C is recommended to lyse cells.

    During thermocycling a 15–30 second denaturation at 94°C is recommended.

  7. Annealing:

    The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm. The NEB TmCalculator is recommended for calculation of an appropriate annealing temperature.


  8. Extension:

    The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.

  9. Cycle Number:

    Generally, 25–35 cycles yield sufficient product. Up to 45 cycles may be required to detect low copy number targets.

  10. 2-step PCR:

    When primers with annealing temperatures of 68°C or above are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.

  11. PCR Product:

    A significant portion of the PCR products generated using OneTaq Quick-Load DNA Polymerase contain dA overhangs at the 3´end; therefore the PCR products can be ligated to dT/dU-overhang vectors.