Protocol for LongAmp™ Taq PCR Kit
The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (2). Taq DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using New England Biolabs’ LongAmp Taq DNA Polymerase. These guidelines cover routine PCR reactions. PCR of templates with high GC content, high secondary structure or low template concentrations may require further optimization.
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94°C).
|Component||25 μl reaction||50μl reaction||Final Concentration|
|5X LongAmp Taq Reaction Buffer||5 µl||10 μl||1X|
|10 mM dNTPs||0.75 µl||1.5 μl||300 µM|
|10 µM Forward Primer||1 µl||2 μl||0.4 µM (0.05–1 µM)|
|10 µM Reverse Primer||1 µl||2 μl||0.4 µM (0.05–1 µM)|
|Template DNA||variable||variable||<1,000 ng|
|LongAmp Taq DNA Polymerase||1 µl||2 µl||5 units/50 µl PCR|
|Nuclease-free water||to 25 µl||to 50 µl|
Notes: Gently mix the reaction. Avoid pipetting samples containing target DNA when amplicons above 20 kb are desired. 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 94°C and begin thermocycling:
Thermocycling conditions for a routine PCR:
|Initial denaturation:||94°C||30 seconds|
- General Guidelines:
The quality of the DNA template is essential for long-range PCR amplification. Recommended amounts of DNA template for a 50 μl reaction is as follows:
DNA up to 15 kb above 15 kb genomic 1 ng–500 ng 10 ng–1 μg plasmid or viral 1 pg–10 ng 10 pg–10 ng
Successful amplification above 20 kb largely depends on the quality of DNA templates and the primer sequences.
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 (https://bioinfo.ut.ee/primer3) can be used to design or analyze primers. For amplicons larger than 20 kb, it is desirable to have primers with GC content above 50%, matched Tm above 60°C, and primers at least 24 nucleotides in length. The final concentration of each primer in a PCR reaction may be 0.05–1 μM, typically 0.1–0.5 μM.
- Mg++ and additives:
Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with LongAmp Taq DNA Polymerase. The final Mg++ concentration in 1X LongAmp Taq Reaction Buffer is 2 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgSO4.
Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (4) or formamide (5).
The recommended final concentration of dNTPs for long-range PCR is 300 μM of each deoxynucleotide.
- LongAmp Taq DNA Polymerase concentration:
We generally recommend using LongAmp Taq DNA Polymerase at a concentration of 100 units/ml (5 units/50 μl reaction). However, the optimal concentration of LongAmp Taq DNA Polymerase may range in specialized applications.
An initial denaturation of 30 seconds at 94°C is sufficient for most amplicons 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 5 minute denaturation at 94°C is recommended. During thermocycling a 10–30 second denaturation at 94°C is recommended.
During thermocycling a 10–30 second denaturation at 94°C is recommended.
The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–65°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.
When primers with annealing temperatures above 60°C are used, a 2-step PCR protocol is possible (see #10).
The recommended extension temperature is 65°C. Extension times are generally 50 seconds per kb. A final extension of 10 minutes at 65°C is recommended.
- Cycle number:
Generally, 25–35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.
- 2-step PCR:
When primers with annealing temperatures above 60°C are used, a 2-step thermocycling protocol is possible.
Thermocycling conditions for a routine 2-step PCR:
STEP TEMP TIME Initial denaturation: 94°C 30 seconds 30 cycles:
- PCR product:
The majority of the PCR products generated using LongAmp Taq DNA Polymerase contain dA overhangs at the 3´–end; therefore the PCR products can be ligated to dT/dU-overhang vectors.