A Primer on Primers

Overview

PCR 

This document will give you an overview on the proper methods of dealing with primers. What to do when they arrive, how to dilute them, and how to store them.

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
5X Phusion HF or GC Buffer	2 µl	10 µl	1X
10 mM dNTPs	0.2 µl	1 µl	200 µM
10 µM Forward Primer	0.5 µl	2.5 µl	0.5 µM
10 µM Reverse Primer	0.5 µl	2.5 µl	0.5 µM
Template DNA	Variable	Variable	< 250 ng
Phusion DNA Polymerase	0.1 µl	0.5 µl	1.0 units/50 µl PCR
Nuclease-free water	to 10 µl	to 50 µl

Notes:

Before adding Taq DNA Polymerase, mix the reaction mixtures (or master mix) well. Once Taq DNA Polymerase has been added, gently mix by slowly pipetting up and down.

Collect all liquid to the bottom of the tube by a quick spin if necessary.

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 using Taq DNA Polymerase

Step	Temperature	Time
Initial Denaturation	98°C	30 seconds
30 cycles: Denaturation Annealing Extension	98°C 45-72°C 72°C	15-30 seconds 15-60 seconds 15-30 seconds/kb
Final Extension	72°C	5 minutes
Hold	4°C	Infinite

General Guidelines: 

1. Template: 
   
   Use of high quality, purified DNA templates greatly enhances the success of PCR:

   DNA	Amount
   Genomic	50 ng-250 ng
   Plasmid	1 pg-10 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 T_m 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 T_m.  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.