Team:British Columbia/Notebook/Protocols/ColonyPCR

UBC iGEM 2015

Colony PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer

Overview

PCR 

The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (1). Taq DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using NEB's Taq DNA Polymerase. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb may require further optimization.

Protocol

Reaction setup: 

Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (95°C). 

Component 10 μl Reaction 50 μl Reaction Final Concentration
10X Standard Taq Reaction Buffer 10 mM dNTPs 10 µM Forward Primer @mdo
2 Jacob Thornton @fat
3 Larry the Bird @twitter
Component 25 μl reaction 50 μl reaction Final Concentration
10X Standard Taq Reaction Buffer 2.5 μl 5 μl 1X
10 mM dNTPs 0.5 µl 1 μl 200 µM
10 µM Forward Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM)
10 µM Reverse Primer 0.5 µl 1 μl 0.2 µM (0.05–1 µM)
Template DNA variable variable <1,000 ng
Taq DNA Polymerase 0.125 µl 0.25 µl 1.25 units/50 µl PCR
Nuclease-free water to 25 µl to 50 µl  
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 from ice to a PCR machine with the block preheated to 95°C and begin thermocycling.

Thermocycling conditions for a routine PCR: 

STEP
TEMP
 TIME
Initial Denaturation
95°C
30 seconds
30 Cycles 95°C
45-68°C
68°C 		15-30 seconds
15-60 seconds
1 minute/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 Primer3 (http://frodo.wi.mit.edu/primer3) can be used to design or analyze primers. The final concentration of each primer in a reaction may be 0.05–1 μM, typically 0.1–0.5 μM.

  3. Mg++ and additives: 

    Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with Taq DNA Polymerase. The final Mg++ concentration in 1X Standard Taq Reaction Buffer is 1.5 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2. 

    Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (3) or formamide (4).

  4. Deoxynucleotides:

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

  5. Taq DNA Polymerase Concentration: 

    We generally recommend using Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 μl reaction). However, the optimal concentration of Taq DNA Polymerase may range from 5–50 units/ml (0.25–2.5 units/50 μl reaction) in specialized applications.

  6. Denaturation: 

    An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95°C is recommended. 

    During thermocycling a 15–30 second denaturation at 95°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 Tm Calculator is recommended to calculate an appropriate annealing temperature.

    When primers with annealing temperatures above 65°C are used, a 2-step PCR protocol is possible (see #10). 

  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 yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.

  10. 2-step PCR: 

    When primers with annealing temperatures above 65°C are used, a 2-step thermocycling protocol is possible.

    Thermocycling conditions for a routine 2-step PCR: 

    STEP
    		 TEMP
    		 TIME
    Initial Denaturation
    		 95°C
    		 30 seconds
    30 Cycles
    		 95°C
    65-68°C
    		 15-30 seconds
    1 minute/kb
    Final Extension
    		 65-68°C
    		 5 minutes
    Hold
    		 4-10°C
    		 
  11. PCR product: 

    The PCR products generated using Taq DNA Polymerase contain dA overhangs at the 3´–end; therefore the PCR products can be ligated to dT/dU-overhang vectors.

References:
1. Saiki R.K. et al. (1985). Science. 230, 1350-1354.
2. Powell, L.M. et al. (1987). Cell. 50, 831-840.
3.  Sun, Y., Hegamyer, G. and Colburn, N. (1993). Biotechniques. 15, 372-374.
4.  Sarkar, G., Kapelner, S. and Sommer, S.S. (1990). Nucleic Acids Res.. 18, 7465.