PCR Using Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515)
Reaction Setup:
Q5U Hot Start High-Fidelity DNA Polymerase is inhibited at room temperature, allowing flexible reaction setup (room temperature or ice).
All components should be mixed prior to use.
Section 1: General PCR, USER®Cloning, dUTP incorporation/Carryover prevention
| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
|---|---|---|---|
|
5X Q5U Reaction Buffer |
5 µl |
10 µl |
1X |
|
10 mM dNTPs |
0.5 µl |
1 µl |
200 µM |
|
10 µM Forward Primer |
1.25 µl |
2.5 µl |
0.5 µM |
|
10 µM Reverse Primer |
1.25 µl |
2.5 µl |
0.5 µM |
|
Template DNA |
variable |
variable |
< 1,000 ng |
|
Q5U Hot Start High-Fidelity DNA Polymerase |
0.25 µl |
0.5 µl |
0.02 U/µl |
|
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 to a PCR machine and begin thermocycling.
Q5U Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.
Thermocycling Conditions for a Routine PCR:
| STEP | TEMP | TIME |
|---|---|---|
|
Initial Denaturation |
98°C |
30 seconds |
|
30 Cycles |
98°C |
5-10 seconds |
|
*55–72°C |
20 seconds |
|
|
72°C |
20-30 seconds/kb |
|
|
Final Extension |
72°C |
5 minutes |
|
Hold |
4–10°C |
*Use of the NEB Tm Calculator is highly recommended.
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 |
|---|---|
|
DNA 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 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.
3. USER DNA Engineering
Target DNA molecules and cloning vector are generated by PCR with 8-12 bases of homology between two fragments. PCR primers start with a 5′ A and contain a single deoxyuracil residue (dU) flanking the 3′ end of the homology region, and can be designed to accommodate multiple-fragment assembly, nucleotide substitutions, insertions and/or deletions. We recommend using the GeneDesign (http://genedesign.thruhere.net/gd/) software to design primers for USER junctions. The best results are typically seen when using each primer at a final concentration of 0.5 µM.
4. Mg++ and additives:
Typically, the Mg++ concentration for Q5U Hot Start High-Fidelity DNA Polymerase should be 2.0 mM. When used at a final concentration of 1X, the Q5U Reaction Buffer provides this optimal Mg++ concentration. The addition of common PCR additives such as DMSO may improve amplification of certain difficult or long targets. In these cases, we recommend the addition of up to 2% DMSO.
5. Deoxynucleotides:
The final concentration of dNTPs is typically 200 μM of each deoxynucleotide.
6. dUTP Incorporation/Carryover Prevention
Q5U Hot Start High-Fidelity DNA Polymerse is a dUTP-tolerant DNA polymerase that efficiently incorporates dUTP and amplifies uracil-containing substrates. To prevent carryover contamination, dUTP and Antartic Thermolabile UDG (NEB#M0372) can be added to the reaction. dTTP can be fully replaced by dUTP in the amplification of certain targets. For best results, we recommend adding dUTP at a final concentration of 200 μM. For UDG activation, a 10 minute, 25°C incubation step should be added before the initial denaturation step. Typical cycling parameters can be used thereafter.
7. Q5U Hot Start High-Fidelity DNA Polymerase concentration:
We generally recommend using Q5U Hot Start High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of Q5U Hot Start High-Fidelity DNA Polymerase may vary from 10–40 units/ml (0.5–2.0 units/50 μl reaction) depending on amplicon length and difficulty. It is rarely helpful to exceed 2.0 units/50 μl reaction, especially for amplicons longer than 5 kb.
8. Buffers:
The 5X Q5U Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. The 5X Q5U Reaction Buffer contains 2.0 mM Mg++ at a final (1X) concentration.
9. Denaturation:
Q5U Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.
An initial denaturation of 30 seconds at 98°C is sufficient for most targets being amplified from pure DNA templates. Longer initial denaturation times can be used (up to 3 minutes) for templates that require it. During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.
10. Annealing:
Optimal annealing temperatures for Q5U Hot Start High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. TheNEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
For high Tm primer pairs, two-step cycling without a separate annealing step can be used (see note 11).
11. Extension:
The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples. Extension time can be increased to 1 minute per kb for long, complex templates, if necessary.
A final extension of 5 minutes at 72°C is recommended.
12. Cycle number:
Generally, 30–35 cycles yield sufficient product. For genomic amplicons, 30 cycles are recommended.
13. 2-step PCR:
When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
14. Amplification of long products:
When amplifying products > 6 kb, it is often helpful to increase the extension time to 1 minute /kb.
15. PCR product:
The PCR products generated using Q5U Hot Start High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5U Hot Start High-Fidelity DNA Polymerase will degrade any overhangs generated.
Addition of an untemplated -dA can be done with Taq DNA Polymerase (NEB #M0267) or Klenow exo– (NEB #M0212).
Section 2: Amplification of bisulfite-converted, deaminated, or damaged DNA (Including FFPE DNA)
| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
|---|---|---|---|
|
5X Q5U Reaction Buffer |
5 µl |
10 µl |
1X |
|
10 mM dNTPs |
0.5 µl |
1 µl |
200 µM |
|
10 µM Forward Primer |
1.25 µl |
2.5 µl |
0.5 µM |
|
10 µM Reverse Primer |
1.25 µl |
2.5 µl |
0.5 µM |
|
Template DNA |
variable |
variable |
< 1,000 ng |
|
Q5U Hot Start High-Fidelity DNA Polymerase |
0.25 µl |
0.5 µl |
0.02 U/µl |
|
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 to a PCR machine and begin thermocycling.
Q5U Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.
Thermocycling Conditions for a bisulfite-converted, deaminated, or damaged DNA:
| STEP | TEMP | TIME |
|---|---|---|
|
Initial Denaturation |
98°C |
30 seconds |
|
35 Cycles |
98°C |
10 seconds |
|
*60–68°C |
20 seconds |
|
|
68°C |
1 minute/kb |
|
|
Final Extension |
68°C |
5 minutes |
|
Hold |
4–10°C |
*Use of the NEB Tm Calculator is highly recommended to determine annealing temperatures. Other Tm’s can be used, but for bisulfite-converted, deaminated, or damaged DNA substrates, this narrower range has been shown to support optimal performance.
General Guidelines:
Template:
Purified DNA templates (where possible) greatly enhances the success of PCR.
Recommended amounts of DNA template for a 50 µl reaction are as follows:
| DNA | AMOUNT |
|---|---|
|
DNA Genomic |
10 ng–1 µg |
17. Primers:
We recommend designing long (~26-35) oligonucleotide primers to amplify bisulfite treated/deaminated DNA. Because bisulfite DNA is often damaged, it is helpful to design targets between 150-500bp. Note that since the DNA strands are no longer complementary after bisulfite-treatment/deamination, an individual primer set will only amplify one strand of the target sequence. The first primer should be designed to anneal to the converted target sequence. The second primer should be designed to anneal to the extension product of the first primer, not the opposite template strand, as would be the case in traditional PCR. Primers with higher annealing temperatures (>60°C as determined by the NEB Tm Calculator) are recommended for optimal performance. If needed, include as many guanines as possible in the priming region or add additional bases to increase the Tm. Longer primers will also increase specificity. Ideally, CpG sites should be avoided; if essential, include them on the 5’-end of the primer and have them synthesized with a mixed base (Y= C/T, R= G/A) at the cytosine position. Computer programs such as MethPrimer, methBLAST or BiSearch can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.
18. Mg++ and additives:
Typically, the Mg++ concentration for Q5U Hot Start High-Fidelity DNA Polymerase should be 2.0 mM. When used at a final concentration of 1X, the Q5U Reaction Buffer provides this optimal Mg++ concentration. The addition of common PCR additives such as DMSO may improve amplification of certain difficult or long targets. In these cases, we recommend the addition of up to 2% DMSO.
19. Deoxynucleotides:
The final concentration of dNTPs is typically 200 μM of each deoxynucleotide.
20. Q5U Hot Start High-Fidelity DNA Polymerase concentration:
We generally recommend using Q5U Hot Start High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of Q5U Hot Start High-Fidelity DNA Polymerase may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. It is rarely helpful to exceed 2 units/50 μl reaction, especially for amplicons longer than 5 kb.
21. Buffers:
The 5X Q5U Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. The 5X Q5U Reaction Buffer contains 2.0 mM Mg++ at a final (1X) concentration.
22. Denaturation:
Q5U Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.
An initial denaturation of 30 seconds at 98°C is sufficient for most targets being amplified from pure DNA templates. Longer initial denaturation times can be used (up to 3 minutes) for templates that require it. During thermocycling, the denaturation step should be kept to a minimum. Typically, a 10 second denaturation at 98°C is recommended for most templates.
23. Annealing:
Optimal annealing temperatures for Q5U Hot Start High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. TheNEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
For high Tm primer pairs, two-step cycling without a separate annealing step can be used (see note 24).
24. Extension:
The preferred extension temperature for bisulfite-converted DNA, deaminated DNA, or damaged DNA is 68°C. Extension times of 1 minute per kb for complex, genomic samples is beneficial when amplifying from challenging, converted and/or damaged substrates. If no amplification occurs, first verify the annealing temperature prior to changing the extension temperature.
A final extension of 5 minutes at 68°C is recommended.
25. Cycle number:
Generally, 35 cycles yield sufficient product. Additional cycles can be added but other optimizations (particularly annealing temperature) should be attempted first.
26. 2-step PCR:
When primers with annealing temperatures ≥ 68°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
27. PCR product:
The PCR products generated using Q5U Hot Start High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5U Hot Start High-Fidelity DNA Polymerase will degrade any overhangs generated.
Addition of an untemplated -dA can be done with Taq DNA Polymerase (NEB #M0267) or Klenow exo– (NEB #M0212).