Reaction components:

1. one-fusion Polymerase
An optimal amount of enzyme in 50µl reaction is 1U. In some cases it could be reduced up to 0.5U per reaction, depending on the length and complicity of amplified DNA sequence. For non-complex amplicons with the length less than 500bp and GC-content <60% amount of HF-Fuzz could be decreased up to 0.5-1.25U per 50mlreaction.

2. Buffer
2.5X Uni Buffers provides very high reproducibility across the wide range of amplification conditions, including “fast-PCR” (reduced time of PCR reaction). 2,5X Uni Buffer contains 1.5mM Mg²+, as the final concentration. In some cases we recommend to optimize Mg concentration in the range 1.5-4.5mM

3. DNTP’s
For most of applications 200mM of each of dNTP’s as final concentration is an optimal. It’s not necessary to optimize dNTP’s concentration. dUTP or other dUTP derivatives should be used replacing TTP in PCR reaction for “anti-contamination” PCR.

4. Primers
Usually 10-20pmol of each specific primer in reaction is enough to get good PCR result. If you are using 2-step PCR with the whole blood as a template, it’s better to use >= 20pmol of each primer.

5. PCR Additives
One-fusion polymerase is compatible with the most of commonly used PCR additives for enhancing of high GC-content DNA templates (glycerol, betaine, DMSO and other). If one will use any additives; take into account the changes of Tm of primers and DNA to correct annealing temperature.

Cycling:

 * optimal: Tm for the primer pair recommended as Tm of the lower primer, for the standard-oligos <20nt.

** For non-complex DNA templates (plasmid DNA, phage DNA, BAC clone) extension time could be reduced up to 15 sec/Kb

1. Denaturation:
a) Initial denaturation for 5 min at 98^oC is necessary only for blood cells lysis;

b) for most applications, including “direct-blood” 5 sec at 98^oC is enough for denaturation of the sample in during PCR run

NOTE: Do not use lower T denaturation, then 98^oC; it can cause problems in PCR (nonspecific amplification, poor yield of PCR product, etc.)

2. Annealing/Extension:

For one-fusion DNA polymerase “Annealing” and “Extension” steps should be combined if:
-Tm of both primers are not differs dramatically (<3^oC);
-Tm of the primers are >65^oC (optimal Tm for the primers lays between 65-70^oC)

If primers Tm is about 60-61^oC for both primers ones can apply simple formula to determine starting Ta/e point - (Tm of the lower primer +72^oC)/2. For most applications, it works fine.

To determine a better Ta/e run gradient amplification.

To avoid nonspecific band formation/smearing during amplification not exceed extension time of 30 seconds and use the highest ramp rate of amplificator ( the ramp rate >4-5^oC preferable)

3. Post-PCR amplicon detection:

-         If you are using whole blood as a template, after amplification spine-down blood cells debris to avoid its application to the gel for DNA detection.

Note: Do not use excess of the blood (>10%), because the post-PCR debris volume of blood cells is very high, and does not allow to run more than 1-2 gels.

Recommendations:
- For complex DNA templates (human DNA) strongly recommended to apply Extension time as 30 sec/Kb for templates > 1, 5 Kb.

- For One-Fusion polymerase Tm of the primers should be corrected, as +3-5^oC, comparing with Taq-based PCR conditions.

- For complex DNA templates (human DNA) strongly recommended to apply extension time as 30 sec/Kb